Isolation of Golgi fractions from colchicine-treated rat liver. II. Electrophoretic characterization.

1. Intact Golgi fractions, three from colchicine- or ethanol-treated rat livers and two from a control, were analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. All the fractions showed very similar electrophoretic profiles with 33 protein bands, some of which, especially albumin, had rather higher density in the secretory vesicle fraction than those in the cisternal fraction. 2. Using albumin as the content marker, the Golgi fractions were subfractionated into membranes and contents by freezing-thawing and sonication followed by centrifugation. Distribution of galactosyltransferase among these membrane preparations showed that this enzyme was more enriched in the Golgi cisternal membranes than in the secretory vesicle membranes. 3. All the membrane preparations from the Golgi complex showed very similar patterns on electrophoresis, which were distinctly different from those of microsomal membranes and of plasma membrane. Furthermore, all the Golgi content subfractions had similar protein components, most of which were also found in serum. The microsomal contents, however, showed a considerably different pattern from those of the Golgi contents. 4. From these results it could be concluded that the secretory vesicles are indeed a member of the Golgi complex despite their different appearance and morphology.     

Isolation and characterization of three different forms of arylamidase from rat skeletal muscle

An arylamidase hydrolysing L-leucine-4-nitroanilide was extracted from rat skeletal muscle homogenate and furified by means of anion-exchange chromatography on DEAE-Sephadex A-50 followed by gel filtration on Sephadex G-150 and Sepharose 6B. The enzyme was isolated in the form of three different protein complexes that differ in molecular weight, kinetic data, and sensitivity to metal ions. As studied by SDS-gel electrophoresis and repeated gel chromatography on Sepharose 6B these forms are: 1. a stable monomer (A1) of M_r 122 000; 2. a stable dimer (A2) of M_r 244 000; and 3. a stable polymer (A3) of more than M_r 4·10⁶. The arylamidase was optimally active at pH 7.3 and did not require metal ions. Treatment with 1,10-phenanthroline resulted in complete inactivation, the activity could be restored by the addition of manganous chloride. The sulphhydryl-blocking reagent 4-hydroxymercuribenzoate strongly inactivated the arylamidase, this inhibition could be reversed by the addition of 2-mercaptoethanol. Addition of phenylmethylsulfonyl fluoride had no effect on the enzyme activity. Furthermore, the influence of metal ions as well as the substrate specificity were investigated and compared for all three forms of arylamidase.     

Incorporation of N-acetylglucosamine from UDP-N-acetylglucosamine into proteins and lipid intermediates in microsomal and golgi membranes from rat liver

Rough and smooth microsomes and Golgi membranes incorporate $\text{N-}\text{acetylglucosamine}$ from $\text{UDP}\text{-N-}\text{acetylglucosamine}$ into endogenous protein acceptors. A lipid intermediate of the dolichol phosphate type participates in this transfer reaction in the case of both microsomal subfractions, but the nature of lipid glycosylation is different in these two fractions. Glucosamine transfer in Golgi membranes does not appear to involve a lipid intermediate. In contrast to the results obtained under in vivo conditions, no glucosamine label is recovered in nascent ribosomal proteins or on luminal secretory proteins after incubation in vitro. Proteolysis of intact vesicles of the subfractions removes glycosylated dolichol phosphate and protein acceptors to various extents and interferes with transferase activities. This finding suggests the possibility that glycosylation at the cytoplasmic side of the membrane of the endoplasmic reticulum may involve a system separate from that acting at the luminal side of the same membrane.     

A possible complementary role of actin microfilaments and microtubules in triacylglycerol secretion by isolated rate hepatocytes

Incubation of isolated rat hepatocytes with phalloidin, cytochalasins (which, respectively, stabilize and destabilize actin microfilaments), or colchicine (which inhibits polymerization of microtubules), resulted in a dose-dependent inhibition of triacyglycerol secretion (an index of very low density lipoprotein secretion). Upon removal of drugs from incubation media, the inhibitory effect of cytochalasin D on triacylglycerol secretion was reversible, while such was not the case for phalloidin. When used at maximal concentrations, the combined presence of phalloidin + colchicine or cytochalasin D + colchicine had additive inhibitory effects upon hepatic triacylglycerol secretion, which was virtually blocked; this was not the case for phalloidin + cytochalasin D. These experiments support the concept that microfilaments and microtubules may have complementary functions for the hepatic secretion of very low density lipoproteins.     

Alterations in labeling of cell-surface glycoproteins from normal and diabetic rat intestinal microvillous membranes

The effect of chronic streptozotocin-induced diabetes was studied on intestinal microvillous membrane surface carbohydrate groups. After 7 weeks of diabetes, purified microvillous membranes were prepared from rat small intestine and surface galactoproteins identified by labeling with galactose oxidase/sodium boro[³H]hydride. Membrane surface sialic acid residues were labeled using the sodium metaperiodate/sodium boro[³H]hydride technique. Membranes were solubilized in SDS and protein labeling analyzed by acrylamide electrophoresis. Membranes from diabetic rats showed an 81% increase in galactoprotein labeling ($\text{P< 0.02}$) while labeling of sialic acid residues was unchanged. The greatest increase in galactoprotein labeling occurred in protein monomers of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 116 000–200 000, where there was a 155% increase in labeling ($\text{P< 0.005}$). These results indicate that intestinal microvillous membrane protein glycosylation is altered in chronic diabetes. This increase in surface membrane carbohydrates could explain the decreased rates of proteolytic degradation previously described for at least one microvillous protein. An increase in membrane galactose groups has also been noted in hepatocyte and kidney glomerular basement membranes, which suggests the presence of a systematic change in membrane protein glycosylation occurring as a result of the diabetic state.     

Differentiation under the control of insulin of rat preadipocytes in primary culture: Isolation of homogeneous cellular fractions by gradient centrifugation

Using a density gradient medium (Percoll) we succeeded in isolating homogeneous cell populations from the stromal-vascular fraction of the inguinal tissue of 3-day-old rats. In primary culture, in medium 199 supplemented with 10% fetal calf serum and 5.5 mM glucose, almost complete differentiation (90%) of these fractions was obtained for the first time in presence of a physiological concentration of insulin (10^?9 M). During the adipose conversion, insulin markedly enhanced the activities of glycerol-3-phosphate dehydrogenase and acid:CoA ligase. When VLDL and heparin were added with insulin to the medium, this effect was not potentiated. On the contrary, VLDL and heparin in presence of insulin increased the triglyceride content of the cells. With VLDL and heparin only, the biochemical and morphological characteristics of the cells were very similar to those observed in control culture. The heavier fraction was morphologically heterogeneous and did not undergo the adipose conversion to the same extent as the two lighter fractions. It was concluded that this model could be helpful in studying the proliferation and the differentiation of preadipocytes at an early stage of development.     

An improved purification and further characterization of the messenger ribonucleic acid for the fatty acid synthetase from rat liver

High purity fatty acid synthetase mRNA has been prepared from rat liver. The translational purity of the mRNA preparation was at least 27% as judged by the percentage of the radioactivity incorporated into acid-insoluble material that was precipitated by anti-fatty acid synthetase antibody. The specific activity of the mRNA was 220-times greater than that reported previously from this laboratory [1]. The large increase in the specific activity was achieved by the repeated use of high resolution linear-log sucrose density gradient centrifugation and the removal of 28 S rRNA by Sepharose 4B chromatography, as well as by the optimization of the K⁺ concentration (160 mM) in the reticulocyte lysate translation system. The mRNA preparation showed a single major band on agarose gel electrophoresis under denaturing conditions, and the translational activity of the fatty acid synthetase mRNA on the gel was found to coincide with this band. The molecular weight of the fatty acid synthetase mRNA is 2.5·10⁶ Da. The mRNA directed the synthesis of fatty acid synthetase with a molecular weight indistinguishable from that of the authentic enzyme subunit (M_r = 240 000). The copurification of the translation product and authentic enzyme revealed that the fatty acid synthetase polypeptides synthesized in the reticulocyte lysate system are assembled in vitro into dimers, the native form of the enzyme.     

Importance of apolipoproteins in lipid metabolism

Lipids, which serve as a source of energy and are an important constituent of cell membrane structure, are readily stored in the body. By definition they are insoluble in water. Specific proteins called apolipoproteins interact with lipids to form soluble lipid-protein complexes called lipoproteins. It is in this form that the major lipids — cholesterol, triglyceride and phospholipid — circulate in plasma. Unesterified fatty acids, another major lipid group, are bound to albumin in the circulation. The plasma lipoproteins are complex macromolecules composed of lipids, apolipoproteins and carbohydrates. The relative proportions of these components differ markedly between lipoprotein classes.

Hyperlipidemia is a term used for increased concentrations of plasma cholesterol and/or triglycerides. Any one plasma lipid is present in several types of lipoproteins. Thus, hyperlipidemia implies the presence of hyperlipoproteinemia. The latter has important therapeutic implications. Most of the recent attempts at classification have been directed at the lipoprotein level of plasma lipid organization.

Decreased concentrations of lipids in plasma can be achieved by altering the rates of metabolism of lipoproteins. Decrease in lipoprotein synthesis, increased catabolism or impaired release from cells into the blood stream may all result in a decrease of plasma lipids. Drugs which affect one or more of these factors are used to treat hyperlipoproteinemia. In order to elucidate the mechanism of action of hypolipidemic drugs it is necessary to understand the lipoprotein defect at the molecular level. This requires a more detailed knowledge of lipoprotein metabolism than is presently available for most of the hyperlipoproteinemias.

This paper will review some of the generally accepted properties of the plasma lipoproteins, describe some difficulties which hamper the understanding of lipoprotein metabolism, and identify possible mechanisms by which drugs may affect lipoprotein metabolism.     

Comparative effects of ethanol, n-propranol and isopropanol on lipid disposal by rat liver.

Besides ethanol, other aliphatic alcohols such as n-propanol and isopropanol induce a triacylglycerol (TAG) accumulation in the liver. To determine whether a common mechanism is responsible for the effects of these three alcohols on hepatic lipid metabolism, each was administered by gastric tube to female Wistar rats at the dose of 50 mmol/kg body wt. Whichever alcohol was administered, the hepatic triacylglycerol accumulation was found to be related to the duration of elevated blood alcohol concentration. After administration of n-propanol or isopropanol, the liver [14C]palmitate uptake was increased whereas hepatic palmitate oxidation to 14CO2 was impaired and palmitate esterification into TAG enhanced; these perturbations were however more discrete than after ethanol administration. In contrast to ethanol and n-propanol which, at the dose presently used, increase precursor incorporation into blood TAG, isopropanol inhibits this incorporation. Interference with the process of very low density lipoprotein (VLDL) synthesis and/or secretion, which appears only at a late stage of isopropanol intoxication, is probably responsible for the intensity and duration of the fatty liver observed after administration of this alcohol.     

The carbohydrate content of apolipoprotein E from human very low density lipoproteins.

The carbohydrate content of the E protein of human very low density lipoprotein (VLDL) was evaluated both by colorimetric methods and by gas liquid chromatography of the trifluoroacetylated 0-methyl glycosides. The major unmodified hexose was noted to be galactose with a mole ratio with respect to protein which ranged from 0.81 to 1.54. N-acetyl glucosamine (molar ratios from 0.52 to 1.76) and N-acetyl galactosamine (molar ratios from 0.73 to 1.59) and the respective unacetylated amino sugars were noted for all of the apoproteins evaluated. Sialic acid (molar ratios from 0.79 to 1.69) was a prominent carbohydrate for each of the E protein preparations. When the apoprotein was exposed to neuraminidase with a resultant loss of two-thirds of the sialic acid, the isoelectric focus behavior was found to be unchanged. The E protein isolated from the very low density lipoproteins of Type III patients (dysbetalipoproteinemia) revealed a carbohydrate content similar to the normals or Type IV patients.     

Isolation of surface lectins of GH3 cells from whole cells and isolated plasma membranes

Lectins localized in the plasma membranes seem to be of special importance for the intercellular interaction mechanisms. We describe the isolation of mannose-binding proteins by Triton X-100 extraction and affinity chromatography on agarose-bound mannose. The isolation procedure was performed with whole GH₃ cells as well as with isolated plasma membranes. For the isolation of plasma membranes of GH₃ cells a mechanical pump was used for the disruption. After differential centrifugation an enriched plasma membrane fraction was achieved by discontinuous sucrose gradient centrifugation. The whole fractionation procedure was controlled by total balance sheets for the marker enzymes of the different cell organelles. The plasma membrane fraction was further characterized by gel electrophoresis and electron microscopy. The SDS gel electrophoresis patterns of the proteins, resulting from the Triton X-100 extraction and the affinity chromatography, are nearly identical for whole cells as well as for the enriched plasma membrane fraction. Therefore we presume these mannose-specific proteins to be plasma membrane bound, showing the molecular properties of integral proteins and having a molecular weight of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 67 000, 57 000, 47 000.     

Cell surface of the fish pathogenic bacterium Aeromonas salmonicida: II. Purification and characterization of a major cell envelope protein related to autoagglutination,adhesion and virulence

The purification of the major protein of the membrane fraction of an autoagglutinating strain of Aeromonas salmonicida is described. This protein, designated as additional cell envelope protein, is water-insoluble, has a molecular weight of about 54 000 and its amino terminal sequence is H₂N-Asp-Val-Leu-Leu. Neither sulphur-containing amino acids nor sugar residues were detected. Its amino acid composition, which shows that the additional cell envelope protein is hydrophobic in nature, is remarkably similar to those of various proteins known to be present in additional surface layers of other bacteria, to the adhesive K88 fimbriae of enteropathogenic Escherichia coli and to a pore protein of the outer membrane of E. coli K12.     

Role of parenchymal and non-parenchymal rat liver cells in the uptake of cholesterolester-labeled serum lipoproteins.

Very low density lipoprotein (VLDL)-remnants, prepared by extrahepatic circulation of VLDL, labeled biosynthetically in the cholesterol (ester) moiety, were injected intravenously into rats in order to determine the relative contribution of parenchymal and non-parenchymal liver cells to the hepatic uptake of VLDL-remnant cholesterol (esters). 82.7% of the injected radioactivity is present in liver, measured 30 min after injection. The non-parenchymal liver cells contain 3.1±0.1 times the amount of radioactivity per mg cell protein as compared to parenchymal cells. The hepatic uptake of biosynthetically labeled (screened) low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterolesters amounts to 26.8% and 24.4% of the injected dose, measured 6 h after injection. The non-parenchymal cells contain 4.3±0.8 and 4.1±0.7 times the amount of radioactivity per mg cell protein as compared to parenchymal cells for LDL and HDL, respectively. It is concluded that in addition to parenchymal cells, the non-parenchymal cells play an important role in the hepatic uptake of cholesterolesters from VLDL-remnants, LDL and HDL.     

mRNA-dependent synthesis of rat apolipoprotein E in vitro: Cotranslational processing and identification of an endoglycosidase H-sensitive glycopeptide intermediate

Total polyadenylated enriched mRNA was prepared from rat liver by guanidine-HCl extraction and oligo(dT)-cellulose chromatography. It was translated $\text{in vitro}$ in an mRNA-dependent wheat germ system and rabbit reticulocyte lysate system, using radiolabeled leucine or methionine as amino acid precursor. A product, designated preapoE, was specifically precipitated by a rabbit anti-rat apoE serum and accounted for 1.5% of the total radioactive peptides. It migrated as a single band of radioactivity on SDS gels with an apparent molecular weight similar to that of mature plasma apoE. Inclusion of dog pancreatic microsomal membranes in the translation reaction resulted in a slightly smaller product (by 500 daltons). It also converted the preapoE from an endoglycosidase H-resistant to an enzyme-sensitive species. This suggests that processing of preapoE takes place by the cotranslational removal of a signal peptide and core glycosylation of the mature protein.     

Cross-linking studies of the protein topography of rat liver microsomes

A cleavable cross-linking reagent, dithiobis(succinimidyl propionate), DSP, was used to study the topography of the proteins in the endoplasmic reticulum membrane of rat liver. Reaction of untreated (control), phenobarbital- or 3-methylcholanthrene-induced microsomes with 0.5 mM DSP for 30 min at 0°C resulted in the cross-linking of a protein with a molecular weight of about 52 000 to form an apparent dimer. In phenobarbital microsomes, a smaller amount of a 52 000-dalton protein also appeared in a dimer in the absence of DSP if N-ethylmaleimide was not included during homogenization. In phenobarbital and 3-methylcholanthrene microsomes, a 48 000-dalton protein was cross-linked by DSP to a protein of about 57 000. In all three types of microsomes, a protein with an M_I of about 52 000 was also cross-linked to a protein of about 79 000. In phenobarbital and control microsomes, cross-linking resulted in an oligomeric protein of approximate molecular weight 180 000 which contained three proteins, two with M_r of about 52 000 and one about 79 000. Under the cross-linking conditions, little or no denaturation of cytochrome P-450 and NADPH-cytochrome c reductase was observed. The aryl hydrocarbon hydroxylase activity was significantly inhibited by the bifunctional cross-linking reagent, DSP, but not by the monofunctional reagent N-succinimidyl-3-(4-hydroxyphenyl) propionate. However, attempts to regenerate the aryl hydrocarbon hydroxylase activity by cleavage of the disulfide linkage with 2-mercapto-ethanol or dithiothreitol were not successful.     

Effects of Ca2+, calmodulin and cyclic AMP on the phosphorylation of endogenous proteins by homogenates of rat islets of Langerhans

Activation of Ca²⁺-calmodulin- and cyclic AMP-dependent protein kinases has been suggested to be involved in stimulus-secretion coupling in the pancreatic β-cell. To study the properties of such kinases and their endogenous protein substrates homogenates of rat islets of Langerhans were incubated with [γ-³²P]ATP. Phosphorylated proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and detected by autoradiography. The phosphorylation of certain proteins could be enhanced by Ca²⁺ plus calmodulin or by cyclic AMP. The major effect of Ca²⁺ and calmodulin was to stimulate the phosphorylation of a protein (P53) of molecular weight 53 100±500 (n = 15). Maximum phosphorylation of protein P53 occurred within 2 min with 2 μM free Ca²⁺ and 0.7 μM calmodulin. Incorporation of label into protein P53 was inhibited by trifluoperazine or W7 but not by cyclic AMP-dependent protein kinase inhibitor. Phosphorylation of a protein of similar molecular weight could be enhanced to a lesser extent in the absence of Ca²⁺ but in the presence of cyclic AMP and 3-isobutylmethylxanthine: this phosphorylation was blocked by cyclic AMP-dependent protein kinase inhibitor. Cyclic AMP also stimulated incorporation of label into polypeptides of molecular weights 55 000 and 70–80 000. The results are consistent with the hypothesis that protein phosphorylation mechanisms may play a role in the regulation of insulin secretion.     

Presence of B-100 in rat mesenteric chyle

Molecular forms of apolipoprotein B (ApoB) were studied in the rat intestinal chyle by SDS-polyacrylamide gel electrophoresis, immunoblotting and immunodiffusion. Time studies on intestinal chyle showed the presence of B-100 in all the samples analyzed within 3 hr after drawing. However, the analyses repeated on day 2 or day 3 revealed disappearance of B-100 and appearance of B-48. Addition of 3 mM EDTA, 10 mM diisopropylfluorophosphate, 5 mM chloroquine and 10 mM epsilon-amino caproic acid slowed down but could not prevent the disappearance of B-100. Chylomicrons isolated from chyle in the presence of preservatives immediately after drawing displayed B-100 as a major and B-48 as a minor ApoB form. However, repeatedly washed chylomicrons or those isolated from chyle 18-24 hr after drawing showed B-48 as the only ApoB present. These results suggest that rat intestine synthesizes B-100 which is quickly converted to smaller molecular form.