Biosynthesis of Lipoprotein by Rat Intestinal Mucosa - The Journal of

12 Pages·2003·1.42 MB·English

THE JOURNALO F I~IOLOGICACLH EMISTRY
Vol. 241, No. 8, Issue of April 25, 1966
I’ririnki in U.S.A.
Biosynthesis of Lipoprotein by Rat Intestinal Mucosa*
(Received for publication, July 13, 1965)
I:IIEUEI~ICK T. HATCH,$ YOSHIRO Aso, LILLIAN M. HAGOPIAN, AND JOEL J. RUBENSTEIN
From the Arteriosclerosis Unit, Medical Xervices, MassachusettsG eneral Hospital, and the Department of Medi-
cine, Harvard Medical School, Boston, Massachusetts
SUMMARY tide maps led us to suggest that lipids in transit become
associated with some of the more abundant soluble proteins
Lymph chylomicrons are at the lipid-rich end of the spec-
of the mucosal cells and that the resulting lipoprotein com-
trum of the lipoproteins. The nature of their protein moiety
plexes appear in the chylomicrons of intestinal lymph.
and its biosynthesis have not yet been fully elucidated.
Thin layer methods of chromatography and electrophoresis
Observations are reported on the incorporation of radioactive
were extensively employed. Their convenience and great
leucine into a “lowest density lipoprotein” by mucosal cells
sensitivity permitted detailed studies to be carried out on
of the small intestine isolated from the rat during absorption
submilligram quantities of material.
of fat. The amino acid incorporation exhibited the char-
acteristic energy requirement and sensitivity to inhibitors of
other systems for biosynthesis of proteins. The protein
moiety was not homogeneous, but appeared to consist of a
small number of species that could be separated by thin
layer electrophoresis in starch granules. Similar, but non-
Lymph chylomicrons appear to contain a small amount of pro-
identical, electrophoretic patterns were obtained with the
tein and thus may be included in the category of “lipoproteins”
residual soluble intracellular protein of the intestinal mucosa
(3-6). The amount of protein present, its identity, and the locus
and with chylomicrons of intestinal lymph.
of its biosynt.hesis continue to be unsettled points (7-10).
The lowest density lipoprotein was isolated from a homog-
There is evidence, first reported by Bragdon in 1958, that the
enate of the mucosal cells. After removal of lipids by ex-
chylomicron protein is at least partly synthesized by the intestine
traction with organic solvents, the protein moiety exhibited
(5). In 1959 Rodbell and Fredrickson (7) and Flodbell, Fred-
several unusual properties in addition to its affinity for lipids:
rickson, and Ono (8) characterized three proteins which were as-
(a) a predominance of apolar amino acids, (b) resistance to
sociated with lymph chylomicrons of the dog and human. One
fractionation procedures involving ionic properties, (c) con-
of these proteins was believed to be identical with the protein
siderable solubility in organic solvents, and (d) peculiarities
moiety of plasma high density lipoprotein. Indirect experiments
of the peptide map that were related to the foregoing features.
have also suggested a relationship between high density lipopro-
Peptide maps were obtained after proteolytic digestion of the
tein and chylomicrons (1 l-13). A second protein may have been
protein moieties of the lowest density lipoprotein fraction,
the same as one found in the very low density (triglyceride-
the residual soluble protein, and lymph chylomicrons by
bearing) fraction of the plasma lipoproteins. These two proteins
means of two-dimensional electrophoresis and chromatog-
were believed to be synthesized by the intestine on the basis of
raphy. Unusual maps were obtained in which most of the
data on incorporation of radioactive amino acids. Except for a
peptide fragments failed to move upon electrophoresis over
single experiment in vitro with isolated mucosal cells of the dog,
a broad pH range, but were readily resolved by chromatog-
contribution of the proteins by the liver through its lymphatic
raphy in organic solvents. Strong, although imperfect,
drainage could not be excluded because the cannulated thoracic
resemblance was noted among the peptide maps of all three
duct probably transported both hepatic and intestinal lymph.
protein fractions. Preliminary studies of low and high
Biosynthesis of the major serum lipoproteins by the liver has been
density lipoproteins of rat serum revealed different, more
well established by experiments in viva and in vitro (14, 15).
complex peptide maps.
More recently, clinical observations in patients with inborn
No definitive conclusion could be drawn about the speci-
errors of lipoprotein metabolism have raised provocative ques-
ficity of the protein which becomes associated with lipid
tions concerning the role of lipoproteins in the intestinal absorp-
during absorption through the intestinal mucosa. A specu-
tion of fat. Patients with Tangier disease, who appear to have a
lative interpretation of the aforementioned similarity in pep-
hereditary deficiency of (or- or high density lipoprotein, have no
* Supported by a grant from the John A. Hartford Foundation, impairment of intestinal fat absorption (16). On the other hand,
Inc. Preliminary reports of this work have appeared (1, 2). the deficiency of P-lipoprotein, which was not implicated in fat
$ Present address, Bio-Medical Research Division, Lawrence absorption by the biochemical experiments cited above, involves
Radiation Laboratory, P.O. Box 808, Livermore, California
a severe impairment in the absorption of triglycerides of long
94551. This research was performed during the tenure of an
Established Investigatorship of the American Heart Association. chain fatty acids (17). The fat appears to enter the intestinal
1655
This is an Open Access article under the CC BY license.
1656 Biosynthesis of Lipoprotein by Rat Intestinal Mucosa Vol. 241, No. 8
TABLE I Methods
Incorporation of ‘T-leucine into lipoproteins by rat intestinal
Animal Preparation--Male Sprague-Dawley rats weighing 150
mucosa cells
g were used for the isotope incorporation experiments in vitro.
Mucosal cells from oil-fed rats were incubated for 2) hours at
Larger animals were used for cannulation of the intestinal lym-
37” in a Dubnoff shaker under a gas phase of 95% 02-5yo COZ.
The incubation medium contained 5 ml of Krebs-Ringer-bicar- phatic channels or for preparation of unlabeled protein in quan-
bonate buffer (pH 7.5) (20), 5 mg of glucose, 3 mg of penicillin, tity. The rats were fasted for 18 hours and then were given pellets
0.05 mg of streptomycin, and 10 pC of L-leucine-14C (specific ac- of chow which had been soaked in olive oil or corn oil. Ninety
tivity, usually 240 mC per mmole). At the end of incubation, 1 minutes a.fter the pellets had been eaten (during active fat ab-
mg of nonradioactive leucine was added. Control vessels were sorption), the rats either were operated upon under pentobarbital
incubated, but label was added at the end, after the carrier leu- anesthesia or were killed by a blow on the head and cervical
tine. Tissue and medium were homogenized and fractionated dislocation.
in the preparative ultracentrifuge (see the text). Collection of Zntestinal Chyle-The chylous main intestinal
lymphatic channel was exposed and cannulated with PE-50 poly-
Flotation conditions Experimental Control
Ultracentrifugal - ethylene tubing.1 After stable lymph flow was established, 35
fraction
II lensity Time Force ‘rotein Sacpteivcitif:i ( ;P ‘rot& Sacptqndcyilf c PC of 14C-labeled Chlorella hydrolysate were administered into
~__ __- ~__ -- -- the stomach, followed by 1 ml of corn oil. At the end of the
g/ml hr xg 7ng cpnth % “g ~Wm/W experiment blood was collected by cardiac puncture and the in-
Lowest density testinal mucosa was removed as described below.
lipoprotein. 1.005 0.5 26,000 0.516 771 0.600 2.7 Preparation of Intestinal Mucosal Cells-The small intestine
Very low dell- was irrigated with cold Ringer’s or NaCl solution, and the duo-
sity lipopro- denum and jejunum were excised. The mucosal cells were ex-
tein*... 1.00: 16 105,000 1.63 88 1.58 2.5 truded onto an ice-cold glass plate by scraping with a spatula
Low density
along the serosal surface (19). This preparation appeared to
lipoprotein,& 1.06: 20 105,000 1.03 20 1.19 0
consist largely of whole cells under phase contrast microscopy.
High density
Incubation of Tissue and Isolation of Lipoproteins-Aliquots of
lipoproteina. 1.21 40 105,000 1.01 21 0.90 0
Residual solu- the cell preparation were incubated as described in Table I.
uble protein. 1.21 Sedim- n ts 3.61 85 3.27 8 After 24 hours of incubation, very little labeled lipoprotein was
present in the medium under any of the conditions tested. Ac-
a In most subsequent experiments these three fractions were cordingly, the medium and tissue together were homogenized in a
combined by proceeding directly to density 1.21 after the first Potter-Elvehjem mortar with a motor-driven Teflon pestle for
fractionation step. about 10 up-and-down cycles. The homogenate (4 ml) was
added to 6-ml ultracentrifuge tubes. Water (2 ml) was layered
mucosa from the lumen l ut to remain largely within the cells, on the top and the tubes were centrifuged in the Spinco prepara-
undergoing no detectatl: t)ransport via lymph or plasma chylo- tive instrument for 30 min at 26,000 X g without refrigeration.
microns (18). The foregoing summary of experimental and clini- The tubes were kept in ice except when in the centrifuge.
cal observations suggests that the intestine synthesizes proteins With a syringe and hypodermic needle, 4 ml were transferred
which have a role in the absorption and circulatory transport of from the bottom of each tube (sedimented cell solids being
fat. However, they leave room for further exploration of the avoided) to another tube for density adjustment. The upper 2
essential protein components of the chylomicron and the sources ml were mixed with lipid material adhering to the tube walls.
from which they arise. Water (4 ml) was layered on top in the original tube, and cen-
We present here observations on the biosynthesis of lipoprotein trifugation was repeated for 1 hour at 64,000 x g at a chamber
carried out by isolated mucosal cells of the rat, and a comparison temperature of 14” to remove a small amount of contaminating
of this protein with that of int,estinal lymph chylomicrons. The higher density protein from the lowest density fraction. The
protein appears to be concerned in the process of fat absorption. tubes were then cut with a Spinco tube slicer to yield a top
However, the nature of this protein differs significantly from that. fraction of approximately 2 ml containing all of the turbid
of the lymph lipoproteins obtained in earlier experiment,s with lipoprotein of lowest density. The bottom fraction of this
whole animals of other species (7-9). washing step was discarded.
To the bottom fraction (4 ml) of the original step were added
EXPERIMENTAL PROCEDURE 1.771 g of solid KBr and 1.45 ml of NaCl solution of density 1.005
(which was 1 mM in EDTA) to adjust the mixture to 6 ml with a
MO,t~ialS
solvent density of 1.21 by the method of Radding and Steinberg
r,-Leucine-‘4C, labeled uniformly or in the carboxyl group, and (14). The KBr was dissolved by mixing, and the tubes were
Chlorella protein hydrolysate, labeled uniformly with 14C, were centrifuged for 40 hours at 114,000 x g at 14”. The tubes were
purchased from New England Nuclear Corporation. Puromy- sliced to give top and bottom fractions of 2 ml and 4 ml, respec-
tin, actinomycin D, and chloramphenicol were gifts from Dr. M. tively.
Lamborg, Dr. A. Aisenberg, and Mr. J. T. Murphy, respectively. Serum and lymph were fractionated by ultracentrifugation in
Crystalline bacterial proteinase (Nagarse) was obtained from an analogous manner.
the Enzyme Development Corporat.ion, New York. Trypsin Measurement of Incorporation of Radioactive Leucine-The top
(trichloracetic acid-treated) and pepsin were products of Worth-
ington. Pronase, chymotr)psin, and ribonucleasc were obtained 1 We are indebted to Dr. M. Hori for skillful performance of the
from Calbiochem. surgical procedure.
Issue of April 25, 1966 Hatch et al. 1657
fractions from the first and second centrifugation steps repre- gentle shaking. No turbidity suggesting bacterial contamination
sented lipoproteins of lowest density (corresponding roughly to was noted.
lymph or serum chylomicrons) and other lipoproteins, respec- The pept,ides released by the enzyme were extracted from the
tively. The second bottom fraction (d > 1.21) represented beads with water, and the solution was concentrated under a
residual soluble cellular proteins. The two top fractions and stream of Nz gas. The peptides were finally obtained in a very
2-ml aliquots of the second bottom fractions were treated with small voIume of water. Any insoluble matter was centrifuged,
10 ml of 10% trichloracetic acid solution (containing 0.1 mg of and the yield of peptides was estimated by the method of Lowry
nonradioactive leucine carrier per ml) and were allowed to stand et al. (21).
overnight at 4”. Peptide Mapping and Localization of Radioactive Peptides-
Protein was determined on the fractions by the method of Thin layers (0.375 to 0.50 mm) of Whatman Chromedia cellulose
Lowry et al. (21). Turbidity and solid KBr, if present, were (without binder, type CC41) were prepared on glass plates
eliminated by centrifugation at 3000 rpm before the samples were (20 x 20 cm) with a Desaga apparatus (Brinkmann Instru-
read in the spectrophotometer. Readings were performed at ments). After brief drying in air, the plates were heated for 30
750 mp, or at 500 rnp for larger amounts of protein. Crystalline min at 105”. An estimated 25 to 100 pg of peptides were applied
bovine serum albumin was used as the standard. to the plates in l-p1 portions with drying in air until the total
Aliquots of the suspension of precipitated proteins containing aliquot was added. The plates were then sprayed with the
1 mg or less of protein were filtered on Millipore filters (diameter buffer for electrophoresis (guarding the sample application zone)
24 mm; pore size, 0.8 p) by the method of Loftfield and Eigner and placed in a cell (E-C Apparatus Corporation, Philadelphia).
(22). The filters were washed with 20 ml of the above trichlor- Wicks of Whatman G F/B glass fiber paper were applied to the
acetic acid solution and then with 15 ml of chloroform-methanol ends of the plate, and a clean glass plate or the cell cover was
(2:l) to remove lipids. The filters were dried in air and placed placed on top, separated by spacers from the thin layer plate.
on the bottom of vials; then 5 ml of scintillation mixture (con- Coolant at 15” was circulated through the lower cooling plate.
taining, per liter of toluene, 5 g of 2,5-diphenyloxazole and 0.3 g A regulated potential of 800 volts (16.5 volts per cm with a cur-
of p-bis-1,2’-(5’-phenyloxazolyl)benzene were added. The rent of approximately 25 ma) was applied for 20 to 40 min.
samples were counted in a Packard liquid scintillation counter. The usual electrophoretic buffer consisted of pyridine-acetic
The results were expressed in terms of counts per min per mg of acid-water (1:10:189) at pH 3.5 (28). In some instances,
protein. The amount of protein was measured as described pyridinium-acetate buffer at pH 6.5 and triethylamine-formate
above because it proved impossible to obtain reproducible meas- buffer at pH 9.5 were used. The volatile buffers were subse-
urements directly upon the Millipore filters. As will be shown quently removed by a stream of air.
below, the amount of protein is overestimated in this way. A After the electrophoretic run had been completed, ascending
rather constant proportion of the material giving the color reac- chromatography was performed in the second dimension in
tion apparently consists of peptides of relatively low molecular I-butanol-acet.ic acid-water (4: 1: 1). When the buffer had evap-
weight, which are soluble in trichloracetic acid and pass through orated, the plates were sprayed with 0.4% ninhydrin in 10%
the Millipore filter but are retarded by Sephadex G-25. The aqueous 2-propanol containing 5% (v/v) collidine, and the color
correction factor for the true protein concentration varies with was developed by warming the plates in a gentle flow of steam
experimental conditions from less than 0.5 to about 0.7, but (29).
would be fairly constant within a group of similar samples. Peptides containing radioactive leucine were located by scrap-
PuriJication of Lipoprotein and Residual Protein on Sephadex ing the cellulose in the ninhydrin-positive spots and appropriate
Columns-Preliminary experiments with thin layer chromatog- blank areas with a razor blade. The samples were transferred to
raphy of lowest density lipoprotein and residual protein on counting vials and overlaid with 5 ml of toluene scintillation
Sephadex G-25 (23) revealed evidence for the presence of low mixture. The net counts per unit of plate area were recorded on
molecular weight peptide material in both fractions (24, 25). It a diagram made from the original plate. Slight quenching by
was considered desirable to eliminate such peptides before treat- ninhydrin-peptide colors was not significant.
ment of the proteins with proteolytic enzymes. Each fraction Total Hydrolysis and Amino Acid Composition of Lipoprotein
was therefore passed through a column 0.9 cm in diameter which Protein-The material of the first, turbid peak from the Sephadex
contained 7 g (dry weight) of Sephadex G-25 (coarse beads) with column treatment of the lowest density fraction was extracted
distilled water as eluent. Fractions were collected manually and with organic solvents by the method of Peterson (9) to remove
observations were made of turbidity, protein or peptide, radio- lipids. The protein residue was transferred to a glass ampoule
activity, and bromide ion (when appropriate) in the eluate. with water and 88% formic acid, and the solvents were removed
Digestion uith Proteolytic Enzymes-The material of the first on a rotary evaporator. For about 1.5 mg of protein, 5 ml of
peak from the Sephadex column was highly turbid (resembling redistilled constant boiling HCl were added. The mixture was
chylomicrons) in the case of the lowest density lipoprotein frac- frozen and evacuated with an oil pump for 5 min; partial t,haw-
tion. The first peak of the residual proteins was optically clear. ing was allowed. The ampoule was then sealed under vacuum
Solutions of these materials, containing 0.5 to 5 mg of protein, and heated in a sand bath at 110” for 40 hours.
were lyophilized on glass beads approximately 75 ,U in diameter The ampoule was opened, and its contents were transferred to
(Prism0 Safety Corporation, Huntingdon, Pennsylvania) in a a tube in which three extractions with 5 ml of hexane were
procedure analogous to that presented by Gustafson (26). carried out for removal of residual lipid. The HCl was removed
Lipids were extracted from the protein-laden beads by shaking on the rotary evaporator and several additions of water were
three times with chloroform-methanol (2: 1, v/v). The beads Iikewise removed. The residue was taken up in a small volume
were then incubated with the protease Nagarse (27) (20 to 50 of water.
pg per mg of protein) in water solution for 18 hours at 37” with A sample of protein hydrolysate was dissolved in citrate buffer
1658 Biosynthesis of Lipoprotein by Rat Intestinal Mucosa Vol. 241, x0. 8
30-
CPM(x IO31
mg Protein 20-
TOTAL
LYMPH 2
COLLECTED
( ml 1
1
HOURS -2 0 2 4 6 8 IO 12 14 16
FIG. 1. Incorporation of amino acids into the ultracentrifugal cated by the arrow. Curve 1 is the time course of specific radio-
lipoprotein fractions of lymph in a rat with cannulated intestinal activitv of lvmoh chvlomicron orotein; Curve 2, verg low density
lymphatic channels. After continuous lymph flow was estab- lipoprotein of lymph” (d < 1.005) ; Cur& 5, low ‘plus- high density
lished during fat absorption, 35 &l of ‘Glabeled ChZoreZZa protein lipoprotein of lymph (d 1.005 to 1.21); Curve 4, residual proteins
hydrolysate were administered into the stomach at the time indi- sedimenting at d 1.21.
TABLE III
TABLE II
Incorporation of IT-leucine into lipoproteins by mucosal cells
Incorporation of ‘V-labeled Chlorella hydrolysate amino acids
from fasted or fed rats
into lymph, serum, and intestinal mucosa fractions in vivo
Rats were fasted for 18 hours and were killed either at once or
Intestinal lymph (1.4 ml) was collected from an operated rat
90 min after eating a pellet of chow soaked in olive oil. Incuba-
between 30 and 60 min after intragastric administration of 35 PC
t.ion conditions were as described in Table I.
of uniformly labeled amino acid mixture. At the end of the col-
lection period, serum and a homogenate of intestinal mucosal Fasted rats Fed rats
cells were obtained and fractionated as indicated below. Pro- -_
Ultracentrifugal fraction
tein content and radioactivity were determined as in - “Methods.” Protein Sapceticvifiitcy Protein Sacpteivcitifyic
Flotation conditions
Ultracentrifugal Protein Specific w ~pm/W w cP+CS
fraction r adioactivity Lowest density lipoprotein (f
Density Tiie FCWe
hour; 26,000 X 9; d = 1.005). 0.318 12,150 0.270 8,560
g/ml hr xg w CP~/W Other lipoprotein (40 hours;
Lymph 105,000 x 9; d = 1.21). 1.36 2,003 1.58 1,710
Chylomicrons 1.005 0.5 26,000 0.115 23,400 Residual soluble protein (sedi-
Very low density.. 1.005 16 105,000 0.117 2,760 ments at d = 1.21). 5.18 8,020 4.53 6,970
Residual soluble
protein 1.21 Sediments 9.89 115
Serum at pH 2.2 and subjected to chromatography on a Beckman/
Chylomicrons. 1.005 0.5 26,000 0.140 3,210 Spinco amino acid analyzer.2 Another sample, equivalent to
Bottom fraction.. 1.005 Sediments 20.0 308 0.1 mg, was applied to a plate of silica gel G (20 X 20 cm; 0.5-mm
Mucosa layer thickness), which had been prepared by drying in air at
Lowest density
room temperature (30). This was subjected to ascending chro-
lipoprotein.. 1.005 0.5 26,000 0.070 9,100
matography in a first dimension solvent of 1-butanol-acetic
Very low density.. 1.005 16 105,000 4.68 70
acid-water (4:l :l) and a second dimension solvent of phenol-
Low and high den-
sity. 1.21 40 105,000 6.82 250 water (75:25) with 20 mg of KCN (31). The amino acid pat-
tern was demonstrated with half-strength ninhydrin spray, and
Residual soluble
protein. 1.21 Sediments 23.9 1,176
- 2 We are indebted to Dr. S. C. Hartman for this analysis.
Issue of April 25, 1966 Hatch et al. 1659
TABLE IV
Effect of inhibitors on leucine incorporation
Incubation conditions were as described in Table I, except for the presence of inhibitory compounds. Lipoprotein fractions were
isolated as indicated in Table II.
Lowest density lipoprotein Other lipoprotein Residual soluble protein
Concentration -
Activity Inhibition Activity Inhibition Activity Inhibition
c?ww % % %
Puromycin
Experiment A 0 28,500 6,570 13,500
0.4 x lo- M 12,170 57 2,350 64 2,320 83
Experiment B 0 771 50 85
0.8 x lo- M 10 99 1.5 97 0 100
Experiment C 0 670 143 229
2 x lo- M 0 100 3 98 17 92
Chloramphenicol
Experiment A 0 529 226 1,042
6 x lo-5 M 696 0 215 5 Gl6 41
Experiment B 0 416 97 323
6 x 1o-5 M 300 28 28 71 146 55
Actinomycin D 0 3,260 590 2,110
2 x 1o-6 M 3,300 0 636 0 2,090 1
1 x 1o-5 M 2,610 20 529 10 1,095 48
Ribonuclease
Experiment A 0 414 26 104
2 fig/ml 310 25 31 0 110 0
Experiment Ba 0 31 136 122
2 fig/ml 12 61 41 70 82 33
2,4-Dinitrophenol
Experiment Aa 0 31 136 122
2 x 1o-4 M 2 93 40 71 31 75
(1 Total counts incorporated are reported for these experiments because protein data were unreliable.
the amino acid spots were transferred separately from the plates chromatography. This was extracted with chloroform-methanol
to small test tubes with as much resolution as possible. The (2: 1) by the method of Albrink (34) ; the purified chloroform
amino acids were estimated in a semiquantitative fashion by layer was concentrated and applied to thin layers of silica gel
carrying out the ninhydrin color reaction according to Moore HR (Brinkmann) on microscope slides according to the method
and Stein (32) and removing the silica gel particles by centrifu- of Vahouny, Borja, and Weersing (35). Development was per-
gation before reading in the spectrophotometer. Standard formed with hexane-ether-acetic acid (90: 10: 1) and chloroform-
amino acid mixtures were treated similarly, and the recoveries methanol-water (70:30: 1). After staining with 12 vapor, com-
of individual acids were used in calculating the composition of parison was made with pure standard lipids.
the protein hydrolysates. Ten amino acids and three pairs (Gly- Ribonucleic acid was estimat,ed on aliquots of the Sephadex-
Ser, Leu-Ile, Phe-Trp) could be resolved by this method (cys- treated lowest density lipoprotein and residual soluble protein
teine and methionine were not tested). Acceptable agreement by the method of Schneider (36) after alkaline hydrolysis (37).
with the amino acid analyzer was obtained.
In a separate experiment, the peptide digestion mixture pre- RESULTS
pared with Nagarse was subjected to thin layer electrophoresis
at pH 4.5 in pyridine-acetic acid-1-butanol-water (1: 1:2:36) Incorporation of Radioactive Amino Acids into
(33). The peptide locations were demonstrated on a marginal Lymph Lipoproteins in Vivo
strip with ninhydrin, and the remaining portions of the cellulose,
The incorporation of the amino acids of uniformly labeled
containing the acidic, neutral, and basic peptides, were separately Chlorella hydrolysate into the ultracentrifugal lipoprotein frac-
scraped from the plate, eluted with 5% aqueous 2-propanol, and tions of the lymph is presented in Fig. 1. During the first 2
subjected to total acid hydrolysis as described above. The hours after intragastric administration of the isotopic mixture,
amino acid mixtures were chromatographed in two dimensions there was a rapid increase in the specific radioactivity of all
and measured after elution. flotatable fractions, with peak levels in the following decreasing
Lipid and Ribonucleie Acid Content-The lowest density lipo- order of magnitude: chylomicrons, d < 1.005 and d 1.005 to
protein from fasted rats was isolated after Sephadex column 1.21. The residual protein fraction fd > 1.21) never reached
1660 Biosynthesis of Lipoprotein by Rat Intestinal Mucosa Vol. 241, No. 8
as high a specific activity, although, of course, its protein con- serum proteins or to persistence of unlabeled chylomicrons formed
tent was much higher than that of the lipoprotein fractions. before administration of the labeled mixture.
In a short term experiment, lymph (1.4 ml) was collected be-
tween 30 and 60 min after administration of the labeled amino Incorporation of Radioactive Leucine into Protein Fractions
acids. At the end of the collection period, serum and a ho- of Intestinal Mucosa Cells In Vitro
mogenate of intestinal mucosal cells were obtained. The results
are presented in Table II. In each tissue the chylomicron pro- Characteristics of System-The incorporation of labeled leucine
tein had the highest specific activity. The high level shown by into mucosal protein proceeded at a constant rate for at least
the lymph chylomicrons in comparison to the mucosa is explained 2 hours. The total amount of incorporation into all fractions
by the collection period, which extended into an earlier phase was variable. Values ranged in different experiments from 0.01
when the mucosa probably possessed a higher radioactivity to 0.5% of the added radioactivity. Incorporation was con-
than when the tissue was removed. The lower level in the sistently higher in 150-g rats than in larger animals.
serum chylomicrons may be due to dilution with unlabeled Glucose was required in the incubation medium when the rats
had been fasted or fed pure olive oil by stomach tube, but was
not required when they had been fed a pellet of oil-soaked chow.
+
The requirements for penicillin and streptomycin were not clear,
but it was felt that their presence helped to keep the incorpora-
tion low in control vessels. These antibiotics were present in
all the incubation experiments reported.
The distribution of incorporated radioactivity in the protein
Origin fractions derived from the successive ultracentrifugations is pre-
sented in Table I. In other experiments the specific activity
of the residual soluble protein fraction (d > 1.21) was higher
than in this case, but the lowest density lipoprotein fraction
nearly always exhibited the highest specific activity.
a b C d Little relationship existed between the incorporation of radio-
active amino acid and the richness in absorbed lipid of the iso-
lated mucosal cells, as estimated by their appearance or by
r
: measurement of triglyceride by a modification of the method of
Van Handel (38). The specific radioactivities of lipoprotein
and residual protein fractions also did not differ greatly when
fed and fasted rats were compared (Table III). It was of in-
terest that, when approximately equal amounts of mucosal
tissue were used from fed or fasted rats, the same amount of
protein was found in t,he ultracentrifugal fraction of lowest
density. However, this fraction was markedly turbid when
i. s derived from fed rats and was optically clear when derived from
5:
fasted rats. Thin layer chromatography on silica gel of a chloro-
form-methanol extract of the lowest density fraction from fasted
rats revealed the presence of cholesterol and cholesterol esters,
triglycerides, and lecithin. Small quantities of diglycerides and
nonesterfied fatty acids were also tentat,ively identified. The
glycerides showed overwhelming predominance in a similar
extract from an oil-fed rat.
The lowest density lipoprotein and residual protein fraction
each contained RNA, approximately 37, of the amount of pro-
0 0 :S eo * . . tein. From 7 to 15% of the protein (Lowry reaction) of lowest
a b c ab c d e density lipoprotein and lymph chylomicrons was soluble in
chloroform-methanol (2 : 1).
FIG. 2. A, thin layer (0.37-mm) electrophoresis on potato E$eet of Inhibitors on Amino Acid Incorporation-Data on the
starch granules in barbital buffer; pH 8.6, P/2 = 0.1, 600 volts, effects of several compounds known to inhibit various aspects of
20 ma, 2 hours. The paper was sprayed with ninhydrin in acetone, cell metabolism are presented in Table IV. The results are
and color development was carried out at room temperature.
consistent with those obtained in certain other mammalian
Samples of 20 ~1 containing approximately 100 rg of protein were
applied. a, lowest density lipoprotein; b, residual soluble pro- systems in which protein biosynthesis occurs within intact cells
tein; c, lymph chylomicrons (lipid-extracted); d, human serum (39, 40). However, no attempt has been made at a thorough
(reference marker). L3, thin layer descending chromatography study of experimental design or inhibitor concentrations.
on DEAE-cellulose in the Desaga B-N chamber. Phosphate
buffer, pH 6.8, 0.02 M, with NaCl gradient increasing to 1 M. a, Studies on Homogeneity of Lipoprotein and Residual
r-globulin; b, lowest density lipoprotein; c, residual soluble
Protein Fractions
protein. C, thin layer descending chromatography on Sephadex
G-100 (fine) in the B-N chamber. Phosphate buffer, pH 7.4,
It would not be expected that the lipoprotein or residual pro-
0.05 M. a, hemoglobin; b, lowest density lipoprotein; c, residual
soluble protein; d, r-globulin; e, thyroglobulin. tein fractions isolated from cellular homogenates in the manner
Issue of April 25, 1966 Hatch et al. 1661
described would have been found to consist of single homogeneous purification by Sephadex column chromatography. The studies
proteins because no techniques of high resolving power were are summarized in Fig. 2.
employed. It was important, however, to learn something The lowest density lipoprotein and the residual soluble protein
about the degree of heterogeneity to provide a basis for inter- exhibited similar behavior in several systems. Thin layer elec-
pretation of the succeeding studies of peptide mapping and amino trophoresiso n starch granulesg ave the best resolutiona vailable
acid composition. In order to minimize the technical complica- (Fig. 2A). The lowest density lipoprotein had one major com-
tions resulting from a large amount of lipid in the lowest density ponent and a minor one which moved only a short distance
lipoprotein, these fractions were isolated from fasted animals toward the cathode (perhapso wing to electroendosmosisa),n d
and were studied by electrophoresis and chromatography after another minor component moving rapidly toward the anode.
FIG. 3. Peptide maps on thin layers of cellulose obtained after stance, the first (horizontal) dimension represents movement
extraction of lipids and digestion with Nagarse. A, lowest den- during electrophoresis at pH 3.5 in pyridine-acetate buffer, and
sity lipoprotein fraction; B, lymph chylomicrons; C, residual the second (verlical) dimension represents movement during
soluble protein fraction sedimenting at d = 1.21. In each in- ascending chromatography in 1-butanol-acetic acid-water (4: 1:l).
1662 Biosynthesis of Lipoprotein by Rat Intestinal Mucosa Vol. 241, No. 8
The residual soluble protein appeared to have the same three tion of molecular weights of 110,000 for lowest density lipoprotein
components, but in nearly equal quantities, plus a minor cathodal and 200,000 for residual soluble protein (42, 43). It is possible
band. Lymph chylomicrons from which lipids had been ex- that the Sephadex zones represent stable aggregates of smaller
tracted revealed two components, one apparently corresponding monomers, since we have observed a reversible aggregation of
to the fast anodal fraction of mucosa and the other having ap- the lowest density fraction (after extraction of lipid) when the
proximately the mobility of the main slow band of the lowest ionic strength was lowered by electrodialysis, and some aggrega-
density lipoprotein. tion with precipitation during storage at 4”.
Thin layer chromatography on DEAE-cellulose (Fig. 2B)
showed a single mobile component (of slightly different mobility) Mapping of Peptides Derived from Proteolytic Digestion of
for the two mucosal fractions. Some of the residual soluble Protein Fractions
protein remained at the origin, possibly corresponding to the
Protein Digestion with Various Enzymes-Digestion of the
prominent anodal band seen in electrophoresis. Column chro-
lowest density lipoprotein and residual soluble protein fractions,
matography of the lowest density lipoprotein on DEAE-Sephadex
after Sephadex column treatment, was carried out principally
A-50 was carried out at pH 8.4 with a gradient of Tris-Cl buffer.
with Nagarse. On the basis of estimation of the peptide products
The protein was eluted in a single peak beginning at a buffer
with the reaction of Lowry et al. (21), this enzyme gave more
concentration of about 0.22 M.
extensive hydrolysis of both fractions than did several other
Disk electrophoresis in acrylamide gel columns (41) was rela-
proteases. The yield of soluble peptides recovered after delipida-
tively ineffective because most of the protein of the mucosal
tion was about 50%; significant mechanical losses occurred in
and lymph lipoproteins remained within the sample gel, ap-
the manipulation of small quantities. Part of the protein was
parently uninfluenced by the applied potential.
an insoluble “core” which was removed by centrifugation.
Thin layer chromatography on Sephadex G-100 (fine) (Fig. 2C)
Further digestion of this “core” with Pronase or Nagarse gave a
showed single zones for the two mucosal fractions. Comparison
small additional yield of soluble peptides with practically the
of their mobilities with those of pure proteins permitted estima-
same peptide map as the original Nagarse digest.
Somewhat smaller yields of soluble peptides were obtained by
TABLE V digestion of the lowest density lipoprotein fraction with trypsin
Amino acid composition of protein moiety of lowest in the presence of urea (7), with or without subsequent addition
density lipoprotein of chymotrypsin. Single experiments were also carried out with
The lowest density lipoprotein fraction was isolated from a pepsin and papain, which gave similar results.
homogenate of rat intestinal mucosa by centrifugation for 3 Behavior of Peptides in Two-dimensional Electrophoresis-
hour at 26,060 X g (solvent density, 1.005). The top fraction in Chromatography-There was a strong resemblance between the
the tube was chromatographed on a column of Sephadex G-25 peptide maps of the lowest density lipoprotein and residual pro-
t.o remove contaminating peptide material. The first chromato-
tein fractions (Fig. 3, A and C). Both maps were unusual in
graphic peak contained 1.48 mg of protein. Lipids were removed
that the majority of the peptides did not move at all in the elec-
by treatment with organic solvents, and the protein was digested
tric field, or moved only a distance consistent with electroendos-
with constant boiling HCl for 40 hours at 105’ in a sealed ampoule.
mosis. The electrophoretic immobility of most of the peptides
Extraction with hexane was then carried out to remove a trace
of residual lipid, and the acid was removed on a rotary evapora- was observed at pH 3.5, 4.5, 6.5, and 9.5. In the second (chro-
tor. The residue was dissolved in 2.2 ml of citrate buffer at pH matographic) dimension, the electrophoretically immobile pep-
2.2, and 1.0 ml was applied to each column of an automatic amino tides were resolved into about seven spots distinguishable on the
acid analyzer. basis of mobility or color after ninhydrin spraying, or both.
None of the material remained adsorbed at the origin. The
electrophoretically mobile peptides were resolved into small
pmoles/ numbers of anodal and cathodal spots at pH 3.5, and their mo-
bility varied appropriately with pH.
i 1p.4ro8t eimn g I The final two-dimensional pattern consisted of a column of
peptides that was resolved only in the chromatographic dimen-
Lysine. _. 0.291 9 Glycine. 0.386 12
Histidine.. 0.110 4 Alanine. . 0.323 IO sion and a small number of peptides that moved in both dimen-
Ammonia. 4.72 Cysteine Trace sions. Thus large regions of the map were devoid of any de-
Arginine. _. 0.220 7 Valine. _. . 0.330 11 tectable peptides. However, many experimental variations of
Asparagine. 0.403 I3 Methionine” 0.100 4 these techniques failed to produce greater resolution of the pep-
Threonine.. 0.257 8 Isoleucine 0.242 8 tide mixture. Under similar conditions, proteolytic digests of
Serine.......... 0.298 10 Leucine. 0.477 15 hemoglobin, ribonuclease, and albumin were well resolved over
Glutamic acid.. 0.450 14 Tyrosine. 0.142 5 all regions of the map.
Proline. 0.235 8 Phenylalanine 0.249 8
Tot.al . 4.51d 146 Amino Acid Composition of Lowest Density Lipoprotein Fraction
0 Protein estimated by method of Lowry et ~2. (21). Total Amino Acid Composition-The amino acid analysis of
b In estimating number of residues, greatest weight was given the lowest density lipoprotein is presented in Table V. The
to eight amino acids which are most stable during acid hydroly-
presence of a significant amount of material tentatively identi-
sis, and allowances were made for unstable or slowly released
fied as hexosamine is noteworthy. The very large amount of
residues.
NH, observed cannot as yet be explained; it is far in excess of
e Sum of methionine and isomeric sulfoxide peaks.
that which could have been bound in amide linkage with the
d Excludes ammonia.
Issue of April 25, 1966 Hatch et al. 1663
dicarboxylic amino acids, but a part may have been derived acids, with only a few others present; (c) about 80% of the total
from degradation of hexosamine. leucine plus isoleucine content was in the neutral peptide frac-
A portion of the foregoing lowest density lipoprotein was tion, with about 10% each in the acidic and basic peptide frac-
carried through the acid hydrolysis and then subjected to two- tions. The incorporation of radioactive leucine was likewise
dimensional partition chromatography on a thin layer of silica limited almost entirely to the neutral peptides.
gel G. Two unidentified ninhydrin-positive spots were noted:
one was between aspartic and glutamic acids and the other w&s Peptide Maps of Rat Lgmph Chylomicrons and Serum Lipoproteins
between histidine and proline. The latter may be hexosamine. Peptide maps were prepared for lymph chylomicrons (Fig. 3B)
Amino Acid Composition of Separated Acidic, Neutral, and and chylomicrons, very low density, low density @I), and high
Basic Peptide Fractions-A sample of 400 pg of peptide digestion density (cyr) lipoproteins of serum (Fig. 4). The pattern ob-
mixture from the lowest density lipoprotein was separated in tained from lymph chylomicrons was nearly, but not quite,
the electrophoretic dimension only at pH 4.5. After a guide identical with that of the lowest density lipoprotein of intestinal
strip at the edge of the plate was colored with ninhydrin, the mucosa.
acidic, neutral, and basic peptide regions were scraped from the When our technique was applied to the serum lipoprotein
plate and eluted from the cellulose. The three fractions were fractions, the results were not so satisfactory nor reproducible
hydrolyzed with HCl, and the amino acid mixtures were analyzed as with the mucosa and lymph. Drawings of the peptide maps
by the twodimensional thin layer technique described. Three for each serum fraction are presented in Fig. 4. The chylomicron
noteworthy features were revealed: (a) the neutral peptides pattern had a column of electrophoretically immobile peptides
contained an essentially complete complement of amino acids; which was complex in appearance (resembling a double column).
(b) 50% of the residues in the acidic peptides were dicarboxylic There were two streaked zones moving toward the anode that
amino acids, with small amounts of several other amino acids, were similar to those of mucosa and lymph, but additional anodal
and 46% of the residues of the basic peptides were basic amino peptides were also present. The very low density lipoprotein
PEPTIDE MAPS OF RAT SERUM LIPOPROTEINS
.-------- ----
iolvent front ---------
0-y
- .-Origin
- Very Low Density Lipoprotein
- +
Low Denstty Ltpoprot.etn r&t1 High Density Lipoprotein (02)
FIG. 4. Peptide maps on thin layers of cellulose obtained after represents movement during ascending chromatography in l-
ultracentrifugal fractionation of rat serum, extraction of lipids, butanol-acetic acid-water (4: 1: 1). Dashed lines, continuous
and digestion with Nagarse. In each map the first (horizontal) lines, and filled outlines represent increasing intensity of ninhydrin
dimension represents movement during electrophoresis at pH 3.5 staining. Peptide colors differing from the usual violet are indi-
in pyridine-acetate buffer, and the second (vertical) dimension cated in two instances.
1664 Biosynthesis of Lipoprotein by Rat Intestinal Mucosa Vol. 241, No. 8
exhibited a simple pattern with over-all resemblance to those of and in the semiquantitative assay of amino acid composition, it
the lowest density fraction and lymph chylomicrons. Close has been feasible to work with amounts ranging between 25 and
examination, however, showed a lack of correspondence of the 100 pg of material. Without the availability of these methods
individual peptides. Low density lipoprotein gave a map with it would have been necessary to pool samples from substantial
much greater complexity in the anodal region. High density numbers of animals, and the procedures would have become much
lipoprotein showed a larger number of individual peptides, with more cumbersome.
greater scatter over the plate, and relatively fewer zones did
not move in the electrophoretic dimension. Acknozoledgments-The authors acknowledge helpful discus-
sions of various aspects of this work with Drs. J. M. Buchanan,
DISCUSSION V. M. Ingram, E. Haber, S. C. Hartman, K. J. Isselbacher, and
The role of the intestine in lipoprotein metabolism is poorly R. B. Loftfield. Dr. G. I’. Canellos assisted in the initial phase
understood. Biosynthesis of protein by the intestinal mucosa of this work, and Miss C. E. Anderson participated during a
appears to be essential for lipid transport, since Sabesin and summer under the Advance Studies in Science Program of
Isselbacher have recently reported that inhibition of protein Thayer Academy and the National Science Foundation. The
synthesis in the rat prevents chylomicron formation and ali- typescript was prepared by Mrs. B. L. Jacobson.
mentary lipemia (44). With regard to the specificity of protein
in the chylomicron, previously reported evidence is not definitive. REFERENCES
On the one hand, it is suggested that specific chylomicron pro-
1. HATCH, F. T., HAGOPIAN, L. M., RUBENSTEIN, J. J., AND
teins may be synthesized by the intestinal mucosa (7, 8) ; on the
CANELLOS, G. P., Circulation, 28, 659 (1963).
other hand, nonspecific adsorption of proteins into chylomicrons 2. HATCH, F. T., Aso, Y., HAGOP~AN, L. M., A& RUBENSTEIN,
from the environment is favored (9). J. J., Abstracts of the 148th Meeting of the American Chemical
In the studies reported here, active biosynthesis of mucosal Society, Chicago, September 1964, p. 43C.
3. LINDGREN, F. T., AND NICHOLS, A. V., Plasma Proteins, 2,
proteins has been demonstrated in viva and in vitro by means of
1 (1960).
the incorporation of labeled amino acids and the effects of in- 4. DOLE, V. P., AND HAMLIN, J. T., Physiol. Rev., 42, 674 (1962).
hibitors. The fraction of highest specific activity was found to 5. BRAGDON. J. H.. J. Lab. Clin. Med.. 62. 564 (1958).
be associated with lipid during its absorption across the intes- 6. SCANU, A:, AND GAGE, I. H., J. Expk Med., iO9, i39 (1959).
tinal mucosa. This protein fraction was not homogeneous, but 7. RODBELL, M., AND FREDRICKSON, D. S., J. Biol. Chem., 234,
562 (1959).
exhibited several unusual properties in addition to its affinity
8. RODBELL, M., FREDRICKSON, 1). S., AND ONO, K., J. Biol.
for lipids: (a) a predominance of apolar amino acids (Table V) Chem., 234, 567 (1959).
similar to that which is present in two other lipoproteins, struc- 9. PETERSON, M. L., Thesis, The Rockefeller Institute, 1960.
tural protein of the mitochondrion, and myelin proteolipid (45) ; 10. ISSELBACHER, K. J., AND BUDZ, D., Nature, 200, 364 (1963).
11. SCANU, A., AND HUGHES, W. L., J. Biol. Chem., 236, 2876
(b) resistance to fractionation procedures involving ionic prop-
(1960).
erties; (c) considerable solubility in organic solvents; and (d) 12. FURMAN, R. H., HOWARD, R. P., LAKSHMI, K., AND NORCIA,
peculiarities of the peptide map that are related to the first L. N., Am. J. Clin. N&r., 9, 73 (1961).
three properties. 13. LEVY, R. I., LEES, R. S., AND FREDRICKSON, D. S., J. Clin.
Invest., 44, 1068 (1965).
Our studies again fall short of settling the dilemma about the
14. RADDING. C. M.. AND STEINBERG. I D.. , J. Clin. Invest.. , 39. ,
specificity of the protein which becomes associated with lipid
1560 (1960).
during transport. Further comment depends almost entirely 15. MARSH, J. B., J. Biol. Chem., 238, 1752 (1963).
upon a speculative interpretation of the starch electrophoretic 16. FREDRICKSON, D. S., ALTROCCHI, P. H., AVIOLI, L. V., GOOD-
patterns and the unusual peptide maps observed for various MAN, D. S., AND GOODMAN, H. C., Ann. Internal Med., 66,
1016 (1961).
fractions. The key to this interpretation is the notion that 17. SALT, H. B., WOLFF, 0. H., LLOYD, J. K., FOSBROOKE, A. S.,
patterns obtained from the residual protein fraction presumably CAMERON, A. H., AND HUBBLE, D. V., Lancet, 2, 325 (1960).
represent the most abundant soluble proteins of the mucosal 18. ISSELBACHER, K. J., SCHEIG, R., PLOTKIN, G. R., AND CAUL-
cells. There is an incomplete but strong resemblance among FIELD, J. B., Medicine, 43, 347 (1964).
19. DAWSON, A. M., AND ISSELBACHER, K. J., J. Clin. Invest.,
the electrophoretic patterns (Fig. 2A) and the peptide maps
39, 150 (1960).
(Fig. 3) of (a) the residual protein fraction, (b) the lowest density 20. UMBREIT, W. W., BURRIS, R. H., AND STAUFFER, J. F., Mano-
lipoprotein, and (c) the intestinal lymph chylomicrons. It is metric techniques, Ed. 3, Burgess Publishing Company,
possible that this resemblance means that lipids in transit through Minneapolis, 1957, n. 149.
21. LOWRY, 0: H.,‘RosEBRouGH, N. J., FARR, A. L., AND RANDALL,
the mucosa become associated with some of the more abundant
R. J.. J. Biol. Chem.. 193. 265 (1951).
soluble proteins of the cells and that the resulting lipoprotein
22. LOFTFIELD, R. B., AND EIGNER,‘E. k., Acta Chem. Stand.,
complex then appears in the intestinal lymph. One would par- 17 (suppl.), 117 (1963).
ticularly wish to understand the relationship between the pro- 23. DETERMANN, H., Ezperientia, 18, 430 (1962).
teins we have described and the endoplasmic reticulum of the 24. BANASZAK, L. J., AND MCDONALD, H. J., Biochemistry, 1, 344
(1962).
mucosa, for this cell organelle is believed to play an important
25. ASHWORTH, L. A. E., AND GREEN, C., Biochem. J., 89, 561
role in intestinal fat absorption (46-48). (1963).
26. GUSTAFSON, A., Biochim. Biophys. Acta, 84, 223 (1964).
Thin Layer Methodology 27. HAGIHARA. B., Enzvmes, 4, 195 (1960).
28 NAUGHTON, M!. A.,SANGER, F., HARTLEY, B. S., AND SHAH,
A brief comment is appropriate on the usefulness of thin layer
D. C., Biochem. J., 77, 149 (1960).
methodology in this work. In the mapping and measurement
29. LEWIS, P. R., Biochem. J., 62, 330 (1952).
of radioactivity of peptides obtained from proteolytic digestion 30. BRENNER, M., NIEDERWIESER, A., AND PATAKI, G., in E.