Stimulation of the biosynthesis of membrane glycoproteins from zajdela ascites hepatoma cells by Robinia lectin

Membrane glycoprotein biosynthesis of ascites hepatoma cells is followed by [¹⁴C]glucosamine and [³H]leucine incorporation into cells in culture. The rate of incorporation is strongly increased by the addition of Robinia lectin in culture medium. Labeled glycoproteins are released from lectin stimulated and non-stimulated ceils by trypsin digestion. Studies of labeled trypsinates on sodium dodecyl sulfate gel electrophoresis and Sephadex G-200 filtration exhibit two fractions both labeled with [¹⁴C]glucosamine and [³H]leucine and having different molecular weights, one over 200 000 and the other about 2000. Identical results are obtained when external membrane glycoproteins are solubilized by sodium deoxycholate. Comparison of surface glycoproteins isolated by trypsinization from control cells labeled with [³H]glucosamine and from lectin stimulated cells labeled with [¹⁴C]glucosamine displays no significant qualitative differences between glycoprotein fractions released from both cell groups.     

Analysis of the pinocytic process in rat kidney II. Biochemical composition of pinocytic vesicles compared to brush border microvilli,lysosomes and basolateral plasma membranes

Pinocytic vesicles, brush border microvilli, lysosomes and basolateral plasma membranes were isolated from rat kidney cortex and their biochemical composition and membrane turnover compared. Pinocytic vesicles are devoid of marker enzymes of brush border microvilli, such as alkaline phosphatase and 5′-nucleotidase, and of lysosomes, such as acid phosphatase and β-glucuronidase. The protein pattern as revealed by polyacrylamide gel electrophoresis differs for all four membranes. Analysis of the phospholipid composition shows that pinocytic vesicles are rich in the negatively charged phospholipid phosphatidylserine and have a low content of sphingomyelin and phosphatidylethanolamine.[¹⁴C]guanido-arginine, [³H]fucose and $\text{myo-}\text{[}{}^3\text{H]inositol}$ were preferentially incorporated into the pinocytic vesicles. Using a double label technique with leucine also, evidence of a more rapid turnover of the pinocytic vesicle membrane proteins was obtained.The results suggest that pinocytic vesicles are not derived from the brush border microvillous membrane but are independent entities that are newly synthesized during the pinocytic process.     

Amphibian intestinal brush border membranes-I. Isolation from Rana catesbeiana tadpole.

1. Intestinal brush border membrane vesicles have been isolated form Rana catesbeiana tadpole. 2. Electron microscopy of brush border membrane vesicles demonstrates a fairly homogenous preparation of vesicles, some of them still containing electron dense material. 3. The dense vesicles probably comprise both microvillus core and membrane. 4. Negative staining of vesicles reveals the presence of knob-like structures (particles) covering the outer surface of the membrane. 5. The membranous fraction is characterized by a high specific activity of alkaline phosphatase, trehalase, glucoamylase, maltase and gamma-glutamyltranspeptidase.     

Stimulation of the biosynthesis of membrane glycoproteins from Zajdela ascites hepatoma cells by Robinia lectin.

Membrane glycoprotein biosynthesis of ascites hepatoma cells is followed by [14C]glucosamine and [3H]leucine incorporation into cells in culture. The rate of incorporation is strongly increased by the addition of Robinia lectin in culture medium. Labeled glycoproteins are released from lectin stimulated and non-stimulated cells by trypsin digestion. Studies of labeled trypsinates on sodium dodecyl sulfate gel electrophoresis and Sephadex G-200 filtration exhibit two fractions both labeled with [14C]glucosamine and [3H]leucine and having different molecular weights, one over 200000 and the other about 2000. Identical results are obtained when external membrane glycoproteins are solubilized by sodium deoxycholate. Comparison of surface glycoproteins isolated by trypsinization from control cells labeled with [3H]-glucosamine and from lectin stimulated cells labeled with [14C]glucosamine displays no significant qualitative differences between glycoprotein fractions released from both cell groups.     

Prednisolone enhances aminopeptidase turnover in adult rat small intestine

In adult male rats, fed prednisolone (0.75 mg/kg/day) for 7 days, brush border aminopeptidase activity was increased (P < 0.001) by 106% compared to pair-fed controls. [¹⁴C]Tyrosine was injected intraperitoneally 16 h and [³H]tyrosine 6 h before death. The ³H/¹⁴C ratio was 1.79 ± 0.21 (S.D.) in purified microvillus membranes from treated rats compared to 1.30 ± 0.16 (P < 0.01) in controls. Polyacrylamide gel electrophoresis of brush border membranes under denaturing conditions showed that the increased double-isotope ratio in membranes from treated rats was mainly in the high molecular weight protein subunits (> 80 kDa) Detergent-solubilized aminopeptidase was purified after in vivo labeling by protein A-Sepharose-antiaminopeptidase affinity chromatography. The ³H/¹⁴C ratio in aminopeptidase was 2.42 ± 0.15 (P < 0.05) in treated rats compared to 1.63 ± 0.13 in controls. Over the experimental period steady-state isotope reutilization and protein labeling was demonstrated and there was no isotope metabolism. Total microvillus membrane lipid content was unaffected by prednisolone. We conclude that prednisolone increases brush border aminopeptidase activity by increasing enzyme turnover. Other high molecular weight brush border proteins were similarly affected.     

SELECTIVE RELEASE OF CONTENT FROM MICROSOMAL VESICLES WITHOUT MEMBRANE DISASSEMBLY : II. Electrophoretic and Immunological Characterization of Microsomal Subfractions

Rough and smooth microsomes were shown to have similar sets of polypeptide chains except for the proteins of ribosomes bound to the rough endoplasmic reticulum (ER). More than 50 species of polypeptides were detected by acrylamide gel electrophoresis, ranging in molecular weight from 10,000 to approximately 200,000 daltons. The content of rough and smooth microsomes was separated from the membrane vesicles using sublytic concentrations of detergents and differential centrifugation. A specific subset of proteins which consisted of approximately 25 polypeptides was characteristic of the microsomal content. Some of these proteins showed high rates of in vivo incorporation of radioactive leucine or glucosamine, but several others incorporated only low levels of radioactivity within short labeling intervals and appeared to be long-term residents of the lumen of the ER. Seven polypeptides in the content subfractions, including serum albumin, contained almost 50% of the leucine radioactivity incorporated during 5 min and cross-reacted with antiserum against rat serum. Almost all microsomal glycoproteins were at least partly released with the microsomal content. Smooth microsomes contained higher levels of albumin than rough microsomes, but after short times of labeling with [³H]leucine the specific activity of albumin in the latter was higher, supporting the notion that newly synthesized serum proteins are transferred from rough to smooth portions of the ER. On the other hand, after labeling for 30 min with [³H]glucosamine, smooth microsomes contained higher levels of radioactivity than rough microsomes. This would be expected if glycosidation of newly synthesized polypeptides proceeds during their transit through ER cisternae. The labeling pattern of membrane proteins in microsomes obtained from animals which received three daily injections of [³H]leucine, the last administered 1 day before sacrifice, followed the intensity of bands stained with Coomassie blue, with a main radioactive peak corresponding to cytochrome P 450. After the long-term labeling procedure most content proteins had low levels of radioactivity; this was especially true of serum proteins which were highly labeled after 30 min.     

Solubilization of brush borders of hamster small intestine and fractionation of some of the components

About 90% of the protein of hamster intestinal brush borders was solubilised in 0.25% (w/v) sodium dodecyl sulphate without total loss of biological activity. Detergent-polyacrylamide gel electrophoresis of the solubilised protein separated 10–15 bands and partially resolved maltase, lactase, sucrase-maltase, trehalase and alkaline phosphatase activities. The disaccharidases, which were associated with the higher molecular weight proteins, were preferentially solubilised with 0.1%. (w/v) Triton X-100, butanol or papain, whereas Tris and NaI extracted only the lower molecular weight proteins, possible derived from the core filaments.Electrophoresis of brush border proteins metabolically labelled with [¹⁴C] glucosamine suggested that many of the membrane-bound enzymes are glycoproteins. However, chromatography of a papain digest on Sephadex G-200 showed that the sucrase-maltase complex can be separated nearly free of carbohydrate without total loss of activity.The importance of characterizing membrane proteins solubilised by a number of techniques is discussed.     

Ketone body transport in renal brush border membrane vesicles

Ketone body uptake by renal brush border vesicles has been investigated. Ketone bodies enter into the brush border vesicles by a carrier-mediated process. The uptake is dependent on an Na⁺ gradient ([Na⁺]outside > [Na⁺]inside) and is electroneutral. The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. A pH gradient (alkaline inside) also stimulates the ketone body uptake. Acetoacetate and 3-hydroxybutyrate share the same carrier as demonstrated by the accelerated exchange diffusion and mutual inhibitory effects.     

Partial purification of the sugar carrier of intestinal brush border membranes. Enrichment of the phlorizin-binding component by selective extractions

Summary The [³H] phlorizin-binding component of brush border vesicles was enrichedin situ by negative purification. Several procedures, known to effect selective solubilization of membrane components, were used separately or in combination to remove proteins unrelated to the binding. Deoxycholate ruptured the vesicles and released 67% of their protein, thereby increasing the specific [³H] phlorizin-binding activity of the pellet three-to fourfold. Extracting the deoxycholate-pellets with either NaI or alkaline solutions released up to 38% of the deoxycholate-insoluble protein without significantly affecting phlorizin binding. The polypeptide composition of the membranes at the different stages was analyzed by NaDodSO₄-polyacrylamide gel electrophoresis. A number of polypeptides present in the original vesicles could be ruled out as essential components of the [³H] phlorizin binding entity.Intact and deoxycholate-treated vesicles were subjected to proteolytic attack. Papain liberated sucrase and isomaltase from intact vesicles, but affected neither other Coomassiestained bands nor phlorizin binding. Neither the protein composition nor the binding properties of sealed vesicles were influenced by trypsin or chymotrypsin. However, all the proteolytic enzymes tested on deoxycholate-treated membranes substantially reduced [³H] phlorizin binding and produced concomitantly the disappearance of several bands from the electrophoretic profile.Pretreatment of vesicles with papain, followed by deoxycholate extraction and incubation in alkaline media, increased the specific binding activity of the membranes up to ninefold by removing close to 90% of the protein. A limited number of polypeptides are suggested as possible candidates for the glycoside-binding site of intestinal brush borders.     

Release of intestinal surface-membrane glycoproteins associated with enzyme activity by brief digestion with papain

Rat intestinal surface-membrane glycoproteins were labelled by intraperitoneal injection of [1-(14)C]glucosamine 4h before the animals were killed. At this time, density-gradient centrifugation of disrupted brush borders indicated that glycoprotein radioactivity was distributed identically with sucrase, a plasma-membrane marker. Labelled brush borders were digested by papain for brief time-intervals known to release surface-enzyme particles without disruption of the unit membrane. Digestion for 5min released 90% of the surface sucrase, and almost one-half of the brush-border glycoprotein and label. On Sepharose 4B column chromatography most of the glycoprotein and label emerged as a single peak. This peak contained the most actively labelled glycoprotein in the brush border and was closely associated with maltase, sucrase, beta-naphthylamidase and alkaline phosphatase. The peak was partially resolved on polyacrylamide-gel electrophoresis into three bands. Each band contained a distinctive enzyme or enzyme pair, and was labelled by [1-(14)C]glucosamine. No periodic acid-Schiff-negative protein was observed in the peak material. Glycoproteins susceptible to brief digestion with papain are therefore closely linked to released surface-enzyme particles. Intestinal surface glycoproteins are heterogeneous with respect to molecular weight, electrophoretic mobility and function.     

Biosynthesis and degradation of gamma-glutamyltranspeptidase of rat kidney

gamma-Glutamyltranspeptidase (gamma GTP) of rat kidney is an intrinsic glycoprotein bound to the plasma membrane and composed of two nonidentical subunits and an amino-terminal portion of the heavy subunit anchors the enzyme to the membrane. The mechanisms of biosynthesis, post-translational processing and degradation of the enzyme were studied using mono-specific antibody raised to gamma-glutamyltranspeptidase purified from rat kidney. The following results were obtained. Double isotope labeling in vivo showed that gamma-glutamyltranspeptidase is synthesized as a precursor form with a single polypeptide chain of 78,000 daltons, and then processed post-translationally by limited proteolysis, resulting in two subunits of 50,000 and 23,000 daltons. Incorporation of [3H]leucine or [35S]methionine into the precursor form increased until 60 min after their intravenous injection, and a pulse-chase experiment showed that the half life of the precursor form was 53 min. [3H]Fucose and [3H]glucosamine could also be incorporated into the precursor form, showing that glycosylation of the enzyme occurs at the stage of the precursor form. Rat kidney labeled with [3H]fucose was subjected to subcellular fractionation. The Golgi fraction contained the glycosylated precursor form and a small amount of subunits, and the plasma membrane fraction contained mostly subunits with a significant amount of precursor, suggesting that post-translational processing of the precursor occurs on the plasma membrane. The apparent half lives of the native enzyme and the heavy and light subunits were all estimated as 4.3 +/- 0.5 days by labeling with [3H]leucine or [3H]fucose. gamma-Glutamyltranspeptidase has a different turnover rate from aminopeptidase M, which is located in the microvillus membrane close to gamma-glutamyltranspeptidase.     

Retinoids increase the incorporation of D-[3H]galactose into epidermal glycoproteins

All-trans retinoic acid increased the incorporation of D-[³H]galactose into particulate and soluble glycoproteins in the epidermis of cultured pig skin slices nearly two-fold. Increased incorporation of D-[³H]galactose was not blocked by tunicamycin. This effect was specific for D-[³H]galactose since the incorporation of D-[³H]glucosamine and L-[¹⁴C]leucine into epidermal glycoproteins was unaffected by all-trans retinoic acid. All-trans retinoic acid and 13-cis retinoic acid had quantitatively similar effects on D-[³H]galactose incorporation. All-trans retinyl acetate and an aromatic retinoic acid analogue (‘Etretinate’) were less effective. SDS polyacrylamide gel electrophoresis and fluorography showed increased incorporation of D-[³H]galactose into all epidermal glycoproteins in the presence of all-trans retinoic acid. There was no evidence for synthesis of new glycoproteins such as mucins.     

Drug-resistant mutants of chinese hamster ovary cells possess an altered cell surface carbohydrate component

Surface label experiments using the galactose oxidase-[³ H] -borohydride technique reveal that cells from drug-resistant Chinese hamster ovary clones possess a surface carbohydrate component of apparent molecular weight 165,000 which is absent from wild-type cells. The component may also be demonstrated by [¹⁴C] glucosamine incorporation but not by [³ H] leucine incorporation or by the lactoperoxidase surface labeling reaction.     

Biochemical characteristics,metabolism, and antitumor activity of several acetylated hexosamines

We have synthesized several potential inhibitors and/or modifiers of the carbohydrate portion of plasma membrane glycoconjugates. These include fluorinated and actylated analogs of D-glucosamine, D-galactosamine, and D-mannosamine. These compounds have been tested to determine their effects on both [¹⁴C] glucosamine and [³H] leucine incorporation into glycoconjugate and on cell growth and viability using P-288 murine lymphoma cells maintained in tissue culture. The most cytotoxic agent tested was 2-acetamido-2-deoxy-1,3,4,6-tetra-O-acetyl-β-D-glucopyranose or simply β-pentaacetylglucosamine which prevented cell growth at 10^?4–10^?3 M. β-Pentaacetylglucosamine cytotoxicity was correlated with its high lipid solubility, having an octanol/water partition coefficient of 0.424 as compared with 0.278 for the β-anomer and 0.017 for N-acetylglucosamine. In vitro metabolism studies with [¹⁴C]-and/or [³H]-labeled pentaacetylglucosamine have indicated intracellular de-O-acetylation leading to the biosynthesis of UDP-N-acetylglucosamine, followed by the incorporation of this sugar into cellular glycoprotein. Concomitant with the formation of increased amounts of this nucleotide sugar, intracellular UTP and CTP pools fell to one third normal within 3 h after the administration of 1 mM pentaacetylglucosamine. At present it is unclear whether the cytotoxicity of β-pentaacetylglucosamine or other similar agents is due to alterations in nucleotide and nucleotide-sugar pools causing a decrease in energy charge and polynucleotide biosynthesis or is due to a direct effect on membrane glycoconjugate biosynthesis.     

The effect of tunicamycin on development of the mammalian embryonic pancreas

The role of cell-surface glycoproteins in histogenesis of the embryonic rat pancreas was investigated by studying the effect of tunicamycin (TM) on in vitro development. TM has been shown to block glycosylation of asparagine residues in glycoproteins by inhibiting formation of dolichol oligosaccharide intermediates. Exposure of Day 15 pancreatic rudiments to 1.0 μg TM/ml for 15 or 24 hr inhibited [³H]mannose, [³H]glucosamine, and [³H]fucose incorporation by 95, 85, and 90%, respectively, while [³H]leucine incorporation was reduced by 35%. Similar results were obtained with Day 17 rudiments. These trends were confirmed using sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and fluorography. Inhibition of [³H]monosaccharide incorporation correlated with reduced binding of RCA I-ferritin conjugates to the cell surface and both effects of TM were reversed by reculturing rudiments in medium lacking the antibiotic. Morphologically, TM treatment resulted in a delay in pancreatic histogenesis and this delay correlated with an inhibition of the normal increase in specific activity of amylase, an acinar cell secretory protein. These effects were not mimicked by treatment with cycloheximide at a concentration which inhibited [³H]leucine incorporation to the same degree observed with TM. The percentage of delayed rudiments decreased as reculturing in the absence of TM was extended.     

Effects of Several Tunicamycin-like Antibiotics on Glycoprotein Biosynthesis in Mung Beans and Suspension-cultured Soybean Cells

The antibiotics Streptovirudin and 24010 were tested to determine their effects on the formation of lipid-linked saccharide intermediates associated with glycoprotein biosynthesis in mung bean (Vigna radiata) and suspension-cultured soybean cells (Glycine max cv. Mandarin). In vitro both compounds strongly inhibited the transfer of N-acetyl[³H]glucosamine from UDP-N-[³H]acetylglucosamine to N-acetylglucosaminyl-pyrophosphoryl-polyisoprenol and lipid-linked oligosaccharides, although they had no apparent effect on the incorporation of [¹⁴C]mannose from GDP-[¹⁴C]mannose into mannosyl-phosphoryl-dolichol with a small inhibition into lipid-linked oligosaccharides. In vivo, Streptovirudin and tunicamycin dramatically inhibited the incorporation of N-[¹⁴C]acetylglucosamine and [³H]mannose into Pronase-released material (glycoproteins), whereas there was no effect on [³H]leucine incorporation into Pronase-released material (protein). Because the action of Streptovirudin and antibiotic 24010 in plants and other systems is similar to that for tunicamycin, these antibiotics are believed to be closely related. The use of tunicamycin is discussed with respect to its importance in studying glycoprotein biosynthesis and function in animal and plant systems.     

Biogenesis of microsomal membrane glycoproteins in rat liver. III. Release of glycoproteins from the Golgi fraction and their transfer to microsomal membranes

The presence in the Golgi fraction of glycoproteins destined to be incorporated into the microsomal membrane was investigated. When incubated in sucrose, washed Golgi vesicles released four major, weakly acidic glycoproteins, some of which could be incorporated into microsomal membranes by incubation. Double labeling with [3H]glucosamine and [14C]leucine demonstrated the incorporation of both protein and oligosaccharide moieties, and the main peak of radioactivity was associated with the 70,000 mol wt region after SDS-gel electrophoresis. The proteins that could be incorporated into microsomes were probably associated to a large extent with the outer surface of the Golgi membrane. Centrifugation of the proteins released from the Golgi in a KBr solution (p = 1.24) resulted in a separation of glycoproteins, those in the top layer most actively incorporated into microsomes. The lipoglycoproteins in the top layer that could be incorporated appeared in the 70,000 mol wt region after SDS-gel electrophoresis, as did the corresponding proteins isolated from the supernate. These results suggest that glycoproteins with completed oligosaccharide chains are released from the Golgi system to the cytosol and are subsequently transferred to microsomes as constitutive membrane components.     

Isolation and characterization of Brush border fragments from mosquito mesenterons

Brush border fragments were isolated from homogenates of mesenterons from the mosquito, Culex tarsalis, by a combination of Ca²⁺ precipitation and differential centrifugation. These preparations were routinely enriched seven- to eightfold for the brush border marker enzyme, leucine aminopeptidase. Alkaline phosphatase, a putative brush border marker for both vertebrate and invertebrate brush borders, was found to be unsuitable for Cx. tarsalis. Isoelectric focusing electrophoresis coupled with histochemical enzyme detection was used to enumerate isozymic species of nonspecific esterases [3], leucine aminopeptidase [1], and alkaline phosphatase [1] in isolated brush border fragments. Leucine aminopeptidase activity was solubilized by papain digestion, suggesting an extrinsic active site for this membrane-bound enzyme. The predominant nonspecific esterase isozyme remained membrane-bound. Conventional staining (ie, Coomassie Blue and silver) of proteins separated by isoelectric focusing, sodium dodecylsulfate, and two-dimensional electrophoresis indicated a simple pattern for brush border fragments, with two proteins predominating among the 11–14 routinely detected.     

Organ culture of suckling rat intestine: Comparative study of various hormones on brush border enzymes

Summary Jejunal mucosa of 6 d-old rats were cultured for 24 and 48 h in the presence of thyroxine, insulin, pentagastrin, glucagon, epidermal growth factor (EGF) or dibutyryl-A-3:5-MP cyclic with or without dexamethasone (DX). The enzymes were assayed on the purified brush borders. The various agents added alone to the basic culture medium had no effect with the exception of DX on the levels of enzyme activities. Dexamethasone alone induced sucrase, stimulated maltase, and protected other brush border enzyme activities (aminopeptidase, lactase, and alkaline phosphatase). When added to DX-supplemented medium, only the following factors modified the levels of enzymatic activities observed with DX alone. Insulin (10⁻⁶ M) increased maltase, alkaline phosphatase, and lactase activity to a greater extent than DX at 24 h culture, the effect being maintained at 48 h on alkaline phosphatase only. At 48 h culture, both EGF (10⁻⁸ M) and dbcAMP (10⁻³ M) decreased DX-induced sucrase activity. The latter agent also depressed DX-stimulated aminopeptidase activity. This work was supported by the Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, and a grant 79.7.1243 from the Délégation Générale a la Recherche Scientifique et Technique. P. M. S. is a recipient of a grant from Fondation de la Recherche Médicale (France).     

Alkaline phosphodiesterase I and alkaline phosphatase I in plasma membranes of herpes simplex virus type 1 transformed hamster cells

Plasma membrane extracts from Herpes simplex virus type 1 transformed hamster embryo fibroblasts were chromatographed on Lens culinaris lectin coupled to Sepharose (LcH-Sepharose) and analysed by dodecyl sulphate polyacrylamide gel electrophoresis. Coomassie blue-staining revealed two major protein bands with apparent molecular weights of 125 000 and of about 75 000–90 000. In plasma membranes isolated from these tumor cells prior labeled with [³H]fucose or [³H]glucosamine these bands contained the highest amounts of incorporated radioactivity. Separation by LeH-Sepharose-affinity chromatography as well as metabolic labeling clearly demonstrates their glycoprotein character. The 125 000 protein coincides with alkaline phosphodiesterase I activity with a K_m of 6 · 10^?4 M for TMP p-nitrophenyl ester and is competitively inhibited by UDP-N-acetylglucosamine. This enzymatic activity is also present in normal hamster embryo fibroblasts. Gel electrophoresis of the Lens culinaris lectin-binding glycoproteins from plasma membranes of normal hamster embryo fibroblasts additionally revealed a strong alkaline phosphatase activity represented by an apparent molecular weight of 150 000, while HSV₁ hamster tumor cells contain only a very weak activity of this enzyme activity. HSV-lytically infected cells, however, have unchanged levels of alkaline phosphatase activity, whereas alkaline phosphodiesterase activity increases slightly.