Subcellular localization of mannosyl transferase and glycoprotein biosynthesis in castor bean endosperm

Differential and sucrose density gradient centrifugation have shown that the mannosyl transferase present in germinating castor bean endosperm cells which catalyses the synthesis of mannosyl-phosphoryl-polyisoprenol is exclusively located in the endoplasmic reticulum membrane. This intracellular location was confirmed using both ribosome-denuded microsomes isolated in the presence of EDTA and rough-surfaced microsomes isolated in the presence of excess Mg²⁺ added to maintain ribosome-membrane attachment. Separation of organelles following the incubation of crude particulate fractions with GDP[¹⁴C]mannose demonstrated that most of the mannolipid thus formed remained associated with the microsomal fraction. When organelles were isolated from intact tissue which had previously been incubated with GDP[¹⁴C]mannose, [¹⁴C]glycoprotein was found to be associated with other cellular fractions in addition to the microsomes, in particular the glyoxysomes. The kinetics of radioactive labelling of these organelles suggest that [¹⁴C]glycoprotein appears initially in the microsomal fraction and subsequently accumulates in the glyoxysomes. Subfractionation of isolated, [¹⁴C]glycoprotein-labelled glyoxysomes established that over 80% of the total radioactivity was present in the membrane, while sodium dodecyl sulphate-polyacrylamide gel electrophoresis of solubilized glyoxysomal membranes showed that the [¹⁴C]sugar moiety was associated with several, but not all, constituent polypeptides.Abbreviations ER endoplasmic reticulum - TCA trichloroacetic acid - SDS sodium dodecylsulphate - GDP guanosine diphosphate     

Incorporation of D-[14C]galactose into organelle glycoprotein in castor bean endosperm

Excised casto bean (Ricinus communis L.) endosperm tissue supplied with [¹⁴C]galactose incorporates radioactivity into particulate cell components. Fractionation of homogenates established that ¹⁴C-labeled trichloroacetic acid-insoluble material was located primarily in the microsomal and glyoxysomal fractions. The capacity of the tissue to incorporate [¹⁴C]galactose into organelle glycoprotein varied during seedling development, increasing during the first 3 days of germination and subsequently declining. The kinetics of incorporation into the major organelle fractions of 2-day old endosperm tissue showed that the endoplasmic reticulum was immediately labeled whereas a lag period preceded the labeling of glyoxysomes. Sub-fractionation of the isolated organelles established that the greatest proportion of the [¹⁴C]-galactose labeled glycoprotein was located in the membrane, although a significant incorporation into the matrix protein was also observed.The results indicate that the addition of the carbohydrate moiety to the polypeptide cores occurs in the endoplasmic reticulum during or immediately after their synthesis on membrane-bound ribosomes.Abbreviations ER endoplasmic reticulum - SDS sodium dodecyl sulphate - TCA trichloroacetic acid     

Glycoprotein biosynthesis in plants. Formation of lipid-linked oligosaccharides of mannose and N-acetylglucosamine by mung bean seedlings.

Cell-free enzyme particles from mung bean seedlings catalyze the incorporation of mannose from GDP-[¹⁴C]mannose and GlcNAc from UDP-[³H]GlcNAc into glycolipids and into glycoprotein. The most rapidly labeled product from GDP-mannose was characterized as a mannosyl-phosphoryl-polyisoprenol, whereas that from UDP-GlcNAc was a mixture of GlcNAc-(pyro)phosphoryl-polyisoprenol and a disaccharide composed of two N-acetylglucosamine residues attached to the polyisoprenol by a phosphoryl or pyrophosphoryl linkage. Radioactivity from GDP-mannose and UDP-GlcNAc was also incorporated into more polar lipids which have been partially characterized as a series of oligosaccharide-(pyro)phosphoryl-lipids. The mannose-labeled oligosaccharides released from these lipids by mild acid hydrolysis were found to contain GlcNAc at their reducing end indicating that these oligosaccharides contain both GlcNAc and mannose. Both the GlcNAc-labeled and the mannose-labeled oligosaccharides gave multiple radioactive peaks upon paper chromatography indicating that they are composed of a series of different sized oligosaccharides. Finally, radioactivity from GDP-[¹⁴C]mannose and UDP-[³H]GlcNAc is incorporated into an insoluble component. Ten percent of the mannose label and all of the GlcNAc label in this insoluble material could be solubilized by digestion with Pronase. The glycopeptides released by Pronase digestion appeared to be approximately the same size as the oligosaccharides from the lipid-linked oligosaccharides based on gel filtration chromatography on Sephadex G-50. The results are consistent with a mechanism for glycoprotein synthesis involving lipid-linked oligosaccharide intermediates.     

Mannosylation of glycoproteins and dolichol derivatives in fibroblasts from patients with cystic fibrosis

Increased incorporation of mannose into endogenous glycoprotein fractions has been found in whole cell lysates and crude membrane preparations of cultured skin fibroblasts from patients with cystic fibrosis (1.3–2.3-times normal) when GDP[¹⁴C]mannose served as the mannosyl donor. In contrast, the incorporation of mannose from GDPmannose into lipid fractions containing dolichol phosphate and dolichol pyrophosphate oligosaccharides as well as the incorporation of mannose from dolichol phospho[³H]mannose into both glycoproteins and dolichol derivatives were not significantly different among cell preparations from patients with cystic fibrosis and normal controls. Mannosyltransferase activity toward exogenous glycoproteins as well as the activities of soluble and membranous α-mannosidase and β-mannosidase appeared to be normal and could not account for the observed differences. The altered incorporation of mannose into endogenous glycoprotein may reflect changes in glycosylation processes other than mannosylation.     

Dolichylphosphate-dependent Glycosyl Transfer Reactions in the Endoplasmic Reticulum of Castor Bean Endosperm

In the endosperm of Ricinus communis (castor bean) a number of glycosyl transferases were found to be present during germination. They catalyze the incorporation of mannose from guanosine diphosphate mannose and of N-acetylglucosamine from uridine diphosphate N-acetylglucosamine into a glycolipid fraction, which had all of the properties of dolichylphosphate and pyrophosphate sugars, respectively. The sugar moiety of dolichylphosphate mannose is transferred to a lipid-oligosaccharide, containing more than 6 hexose units. When the membranes are preincubated with nonradioactive guanosine diphosphate mannose and uridine diphosphate N-acetylglucosamine, radioactivity from dolichylphosphate [¹⁴C]mannose is also transferred to a glycopolymer. In addition, the formation of radioactive glycoproteins from guanosine diphosphate [¹⁴C]mannose has been demonstrated using a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography.     

A lipid-linked oligosaccharide intermediate in glycoprotein synthesis. Characterization of [Man-14C]glycoproteins labeled from [Man-14C]oligosaccharide-lipid and GDP-[14C]Man.

Endogenous proteins of cell-free preparations of hen oviduct labeled from GDP-[14C]Man or from [Man-14C]oligosaccharide-lipid have been compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Under the conditions tested, a polypeptide chain of molecular weight about 25,000 was the principle acceptor for the oligosaccharide moiety of exogenous [Man-14C]oligosaccharide-lipid. The product labeled by [Man-14C]oligosaccharide-lipid appeared identical with one of three glycoproteins formed when GDP-[14C]Man was incubated with a crude membrane fraction. These three proteins (apparent molecular weight of 75,000, 55,000, and 25,000) accounted for nearly two-thirds of the [14C]mannose-labeled glycoprotein products using GDP-[14C]Man and either the crude membrane fraction or a total oviduct homogenate. Thus, all of the mannose acceptor proteins present in the oviduct homogenate appear to be membrane-bound. Analyses of the [Man-14C]glycoproteins labeled from GDP-[14C]Man in membrane fractions from hen kidney, liver, brain, and oviduct indicated that a labeled polypeptide of apparent molecular weight 25,000 was the only major protein product common to the four preparations.     

The transfer of mannose from guanosine diphosphate mannose to dolichol phosphate and protein by pig liver endoplasmic reticulum

When pig liver microsomal preparations were incubated with GDP-[¹⁴C]mannose, 10–40% of the ¹⁴C was transferred to mannolipid and 1–3% to mannoprotein. The transfer to mannolipid was readily reversible and GDP was one of the products of the reaction. It was possible to reverse the reaction by adding excess of GDP and to show the incorporation of [¹⁴C]GDP into GDP-mannose. When excess of unlabelled GDP-mannose was added to a partially completed incubation there was a rapid transfer back of [¹⁴C]mannose from the mannolipid to GDP-mannose. The other product of the reaction, the mannolipid, had the properties of a prenol phosphate mannose. This was illustrated by its lability to dilute acid but stability to dilute alkali, and by its chromatographic properties. Dolichol phosphate stimulated the incorporation of [¹⁴C]mannose into both mannolipid and into protein, although the former effect was larger and more consistent than the latter. The incorporation of exogenous [³H]dolichol phosphate into the mannolipid, and its release, accompanied by mannose, on treatment of the mannolipid with dilute acid, confirmed that exogenous dolichol phosphate can act as an acceptor of mannose in this system. It was shown that other exogenous polyprenol phosphates (but not farnesol phosphate or cetyl phosphate) can substitute for dolichol phosphate in this respect but that they are much less efficient than dolichol phosphate in stimulating the transfer of mannose to protein. Since pig liver contained substances with the chromatographic properties of both dolichol phosphate and dolichol phosphate mannose, which caused an increase in transfer of [¹⁴C]mannose from GDP-[¹⁴C]mannose to mannolipid, it was concluded that endogenous dolichol phosphate acts as an acceptor of mannose in the microsomal preparation. The results indicate that the mannolipid is an intermediate in the transfer of mannose from GDP-mannose to protein. Some 4% of the mannose of a sample of mannolipid added to an incubation was transferred to protein. A scheme is proposed to explain the variations with time in the production of radioactive mannolipid, mannoprotein, mannose 1-phosphate and mannose from GDP-[¹⁴C]mannose that takes account of the above observations. ATP, ADP, UTP, GDP, ADP-glucose and UDP-glucose markedly inhibited the transfer of mannose to the mannolipid.     

Mannosyl transferases inSaccharomyces cerevisiae: Evidence for the occurrence of ectomannosyltransferase activity

The subcellular distribution of mannosyltransferases inSaccharomyces cerevisiae was studied following the separation of the plasma membrane from other intracellular membranous systems. Most of the activity was linked to internal membranes, and the rest was located at the level of the plasma membrane. Yeast plasma membranes coated on their external face with concanavalin A when incubated with GDP-[U-¹⁴C]mannose incorporated 20% less [U-¹⁴C]mannose in glycoproteins and 110% more in glycolipids than plasma membranes alone. This suggested that part of the total mannosyltransferase activity of the plasma membrane is located on its outer surface. A significant incorporation of radioactive mannose into trichloroacetic-acid-precipitable material was detected in incubations of protoplasts with GDP-[U-¹⁴C]mannose when incorporation of free mannose did not occur. Characterization of a product synthesized by the ectotransferase(s) was achieved after treatment of the radioactive plasma membranes by Triton X-100, which preserved the concanavalin A-mannoprotein complexes and removed a large amount of other plasma membrane components. A radioactive glycoprotein band with an apparent molecular weight of 94, 000 was identified as a product of the ectomannosyltransferase(s).     

Amphomycin inhibits the incorporation of mannose and GlcNAc into lipid-linked saccharides by aorta extracts

Amphomycin inhibits the incorporation of mannose from GDP-[¹⁴C]mannose and GlcNac from UDP-[³H]GlcNAc into lipid-linked saccharides by either a particulate or a solubilized enzyme fraction from pig aorta. The solubilized enzyme was much more sensitive to the antibiotic than was the particulate fraction with 50% inhibition being observed at 8–15 μg of amphomycin. Although the antibiotic inhibited mannose transfer from GDP-[¹⁴C]mannose into mannosyl-phosphoryl-dolichol, lipid-linked oligosaccharides and glycoprotein, the synthesis of mannosyl-phosphoryl-dolichol was much more sensitive to amphomycin. Amphomycin also inhibited the incorporation of mannose from GDP-[¹⁴C]mannose into mannosyl-phosphoryldecaprenol in particulate extracts of $\text{Mycobacterium smegmatis}$.     

Glycoprotein synthesis in plants: I. Role of lipid intermediates

The enzymic processes involved in glycoprotein synthesis have been studied using crude extracts obtained from developing cotyledons of Phaseolus vulgaris harvested at the time of active deposition of vicilin. Radioactivity from GDP-[¹⁴C]mannose can be incorporated by crude extracts into a single chloroform-methanol-soluble product as well as into insoluble product(s). Mannose is the sole ¹⁴C-labeled constituent of the lipid. The kinetics of incorporation of ¹⁴C, as determined by pulse and pulse-chase experiments using GDP-[¹⁴C]mannose, as well as direct incorporation from added [¹⁴C]mannolipid, shows that the mannolipid is an intermediate in the synthesis of the insoluble product(s). The characteristics of the mannolipid are consistent with it being a mannosyl phosphoryl polyprenol. The mannose is apparently attached to the lipid via a monophosphate linkage. Of the radioactivity in the insoluble product(s), about 20% is pronase-digestible during a “pulse experiment.” After a chase with unlabeled GDP-mannose, about 40% is pronase-digestible; the other 60% is as yet uncharacterized. A radioactive product soluble in a mixture of chloroform-methanol-H₂O can be extracted from the insoluble residue obtained during a pulse, but is no longer present after a chase. This product may be a lipid oligosaccharide, the final intermediate in glycoprotein synthesis. Data are presented on incorporation from UDP-N-[¹⁴C]acetylglucosamine into both chloroform-methanol-soluble and -insoluble product(s). The results are consistent with an involvement of lipid intermediates in the glycosylation of protein in this system, and support the concept that the mechanisms of glycoprotein synthesis in higher plants are similar to those which have been reported for mammalian systems.     

Polyprenol phosphates and mannosyl transferases in Phaseolus aureus

The transfer of mannose from GDP[¹⁴C]mannose to lipid and to insoluble polymer by a particulate preparation of Phaseolus aureus has been investigated. The evidence favours the lipid being a prenol phosphate mannose. Of a range of prenol phosphates tried, betulaprenol phosphate was the most effective exogenous acceptor of mannose. Most of the insoluble [¹⁴C]polymer formed was glycoprotein in nature although small quantities of ¹⁴C were associated with glucomannan and galactoglucomannan fractions. Time studies failed to reveal a typical precursor-product relationship between the lipid and polymer fractions but on incubation of [¹⁴C]mannolipid with the particulate fraction a small transfer (0·5–0·7%) of [¹⁴C] to polymer was detected. p-Hydroxymercuribenzoate inhibited (by 90%) the transfer of [¹⁴C] from GDP[¹⁴C]-mannoseto polymer and simultaneously increased (3-fold) the [¹⁴C] recovered in the lipid fraction. The effect was nullified by mercaptoethanol. Attempts to solubilize the transfer system were only partially successful. The formation of a chromatographically identical mannolipid was demonstrated in particulate fractions of Codium fragile and tomato roots.     

Elicitor induction of the synthesis of a novel lectin-like arabinosylated hydroxyproline-rich glycoprotein in suspension cultures of Phaseolus vulgaris L.

A novel lectin-like glycoprotein which accumulates in response to fungal elicitor action has been characterised in endomembranes from suspension cultures of French bean (Phaseolus vulgaris L.). The lectin, which has specificity towards N-acetylglucosamine oligomers, consists of a polypeptide of apparent molecular weight (M_r) 31 000 which is rich in glycine and contains 6.7% hydroxyproline O-linked to arabinose-containing oligosaccharides to give a glycoprotein of M_r 42500. A dual-labelling technique has been used to identify changes in the synthesis of the glycoprotein in cells exposed to fungal elicitor molecules. Thus, incorporation of [¹⁴C]proline into membranes in vivo and of [1^-3H]arabinose from uridine 5-diphosphate [1^-3H]arabinose in vitro and analysis by isoelectric focussing-polyacrylamide gel electrophoresis gave absolute correspondence of the labelled isoform of the glycoprotein. Having established the absence of contaminating polypeptides, subsequent analysis of microsomal fractions bysodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the peak of sythesis of the M_r-42500 glycoprotein occurred 4 h after the addition of fungal elicitor. The changes in the level of incorporation into the glycoprotein monomers were concomitant with increases in the activity of prolyl hydroxylase (EC 1.14.11.2)Incorporation of [¹⁴C]proline and its subsequent post-translational modification to hydroxyproline in microsomal polypeptides was followed by rapid transfer into the wall with an average t _1/2 of about 7 min. The M_r-42500 glycoprotein was rapidly transferred out of the endomembrane fraction with a t _1/2 of 2 min and could be detected in wall fractions where it became progressively less extractable. The glycoprotein, which clearly differs from bean extensin, accounts for up to 40% of the hydroxyproline newly exported in response to elicitor action. The lectin, which resembles those found in the Solanaceae and which is coinduced with enzymes of phytoalexin synthesis, may play some role in disease resistance.Abbreviations HRGP hydroxyproline-rich glycoprotein - IEF isoelectric focussing - M_r apparent molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

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.     

Synthesis of salt-soluble proteins in barley. Pulse-labeling study of grain filling in liquid-cultured detached spikes

The accumulation of salt-soluble proteins in the endosperm of developing barley (Hordeum vulgare L.) grains was examined. Detached spikes of barley were cultured at different levels of nitrogen nutrition and pulse-labeled with [¹⁴C] sucrose at specific times after anthesis. Proteins were extracted from isolated endosperms and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. Fluorography revealed an early, middle and late synthesis of specific proteins during grain filling. Synthesis of proteins appearing at the later stages responded to increased nitrogen nutrition. Two major components, -amylase and protein Z in particular, had a synthesis profile almost identical to that of the endosperm storage protein, hordein.Abbreviations CIE Crossed immunoelectrophoresis - SDSPAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis     

The identification of polypeptides synthesised during the acquisition of teichoic acid synthetic activity in Bacillus licheniformis

An attempt has been made to identify proteins synthesised during induction of teichoic acid synthesis in Bacillus licheniformis ATCC 9945. The proteins are recognised as those produced on the change from teichuronic acid to teichoic acid synthesis that occurs after the transfer of the bacteria from phosphate-limited to phosphate-rich conditions. B. licheniformis was grown in phosphate-limiting conditions in the presence of threonine to stimulate threonine uptake. The bacteria were then transferred to phosphate-rich conditions and were pulsed-labelled with [¹⁴C]threonine during the change to teichoic acid synthesis. All of the proteins were extracted from the cells with sodium dodecyl sulphate and were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Radioactive polypeptides were identified by fluorography of the polyacrylamide gels. The radioactive polypeptides that were formed on change from teichuronic acid to teichoic acid synthesis were compared with the polypeptides present in a membrane sub-fraction that had high teichoic acid-synthesising activity. The labelling of nine polypeptides with [¹⁴C]threonine was dependent on new RNA synthesis. Of these nine polypeptides, five were also present in the membrane sub-fraction with the highest teichoic acid-synthesising activity.     

Cell wall regeneration and galactomannan biosynthesis in protoplasts from carob

Protoplast isolation from endosperms of developing carob (Ceratonia siliqua L.) seeds is reported for the first time. These protoplasts regenerated cell walls within 12 h. In order to assess their potential for galactomannan biosynthesis, the incorporation of radioactivity in the regenerated cell wall polysaccharides (CWP) and extracellular polysaccharides (ECP), after feeding these protoplasts with D-[U-¹⁴C]glucose or D-[U-¹⁴C]mannose was studied. The pattern of the radioactive label distribution in the neutral sugars of the trifluoroacetic acid (TFA) hydrolysate of CWP was different from that of the ECP. In the TFA hydrolysis products of the CWP, immediately after protoplast isolation, the greatest level of radioactivity (approximately 90%) was detected in glucose, galactose and mannose. After 2 days protoplast culture, the label in mannose increased. In contrast, immediately after protoplast isolation, approximately 90% of radioactivity of the ECP was detected in galactose and mannose. However, during culture, the radioactivity incorporation in mannose dropped to one third, while that in galactose and arabinose increased significantly. Hydrolysis of the CWP and ECP with -galactosidase and endo--mannanase confirmed that, at least part of mannose and galactose belonged to galactomannan molecules. These results were compared with those obtained upon feeding developing endosperm tissue with D-[U-¹⁴C]mannose. From our results we concluded that protoplasts from endosperm tissues of developing carob seeds, retained the ability of their original explant to synthesize galactomannan, making protoplasts candidates for the study of galactomannan biosynthesis.     

GLYCOPROTEINS AND GLYCOLIPIDS OF SUBCELLULAR FRACTIONS FROM BOVINE RETINA. INCORPORATION OF MANNOSE AND SIALIC ACID1

Abstract— Endogenous lipids and proteins of bovine retina subcellular fractions were labelled from CMP-[³H]NeuNAc and GDP-[¹⁴C]mannose. The bulk of NeuNAc and mannose transfer activity was in membranes other than those from the rod outer segment (ROS). Lighter and heavier membranes, obtained from ROS free membranes by density gradient centrifugation, were the most active for the incorporation of NeuNAc and mannose, respectively. NeuNAc bound to a lipid indistinguishable from gangliosides, and a lipid that contains mannose (mannolipid-I) were found in the fraction extractable with chloroform-methanol (2:1, v/v). Mannose was also incorporated into a lipid fraction extractable with chloroform-methanol-water (1:1:0.3, by vol) (mannolipid-II). Mannolipid-I and mannolipid-II were labile to mild acid hydrolysis. In the presence of ROS free membranes, radioactivity of mannoli-pid-I was transferred to mannolipid-II and from this to proteins. Analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, the proteins labelled from GDP-mannose migrated as a broad peak covering the range of molecular weights 20,000–30,000 and including the zone of rhodopsin migration. The proteins labelled from CMP-NeuNAc showed four radioactive peaks that were coincident with three out of four periodic acid-Schiff (PAS) positive bands.     

ADP-ribosylation reactions in Sulfolobus solfataricus,a thermoacidophilic archaeon

An ADP-ribosylating system was detected in a crude homogenate from Sulfolobus solfataricus, a thermophilic archaeon, optimally growing at 87°C. The archaeal ADP-ribosylation reaction was time-, temperature- and NAD-dependent. It proved to be highly thermostable, with about 30% decrease of ¹⁴C incorporation from [¹⁴C]NAD on incubation at 80°C for up to 24 h. The main reaction product was found to be mono-ADP-ribose. Testing both [adenine- ¹⁴C(U)]NAD and [adenine- ¹⁴C(U)]ADPR as substrates, it was found that acceptor proteins were modified by ADP-ribose both enzymatically, via ADP-ribosylating enzymes, and via chemical attachment of free ADP-ribose, likely produced by NAD glycohydrolase activity. The synthesis of ADP-ribose-protein complexes was shown to involve mainly acceptors with molecular masses in the 40–100 kDa range, as determined by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulphate.     

Serological and developmental relationships between endoplasmic reticulum and glyoxysomal proteins of castor bean endosperm

Glyoxysomes isolated from the endosperm of castor bean (Ricinus communis L.) by sucrose density gradient centrifugation were fractionated into their matrix protein and membrane components. Antisera were raised in rabbits against both the matrix proteins and sodium dodecyl sulphate (SDS)-solubilized membrane proteins. SDS-polyacrylamide gel electrophoresis (PAGE) analysis established that such antisera precipitate all major polypeptide components present in their respective glyoxysomal mixedantigen preparations. Furthermore, when soluble constituents recovered from the microsomal vesicles or solubilized microsomal membranes were challenged with the appropriate glyoxysomal antiserum, serological determinants were again found to be present. Intact endosperm tissue was incubated with [³⁵S]methionine and the kinetics of ³⁵S-incorporation into protein recovered in immunoprecipitates when the glyoxysomal matrix fraction or the soluble fraction released from the microsomes were incubated with anti-glyoxysomal matrix serum were followed. [³⁵S]antigens rapidly appeared in the microsomal fraction whereas a lag period preceded their appearance in glyoxysomes. Interupting such kinetic experiments by the addition of an excess of unlabelled methionine resulted in a rapid decrease in the microsomal content of [³⁵S]antigens and a concomitant increase in glyoxysomal content.Abbreviations SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - ER endoplasmic reticulum     

Isolation ofn-ethylmaleimide-labelled phlorizin-sensitived-glucose binding protein of brush border membrane from rat kidney cortex

A glucose receptor with high affinity for phlorizin from isolated brush border of rat kidney was labelled specifically withN-[¹⁴C]ethylmaleimide and then extracted from the membranes.After the solubilization of the brush borders with sodium dodecyl sulphate theN-[¹⁴C]ethylmaleimide-labelled receptor protein was isolated and was found to have a molecular weight of approximately 30 000 as determined by sodium dodecyl sulphate-polyacrylamide gel disc electrophoresis. The receptor protein eluted from the sodium dodecyl sulphate-containing gels migrates as a single band on sodium dodecyl sulphate-free polyacrylamide gels.The receptor protein can also be released from the brush borders with low concentrations of sodium deoxycholate. Under these conditions the molecular weight of theN-[¹⁴C]ethylmaleimide-labelled receptor protein is approximately 60 000 in contrast to the protein component solubilized with sodium dodecyl sulphate. Since this detergent is known to dissociate the brush border membrane into its protein components, our results suggest that the phlorizin- sensitive glucose receptor protein has a molecular weight of about 30 000.