Secretion of proteins by cultured bovine oviducts collected from estrus through early diestrus

To characterize in vitro protein secretion by the oviduct throughout early stages of the estrous cycle, 16 cows received a luteolytic dose of PGF2 alpha and were randomly assigned to be killed on day (D) 0, 2, 5, or 8 after subsequent estrus. Explant cultures of oviducts (ampulla and isthmus) were incubated for 24 h at 39 degrees C in a modified Eagle's minimal essential medium supplemented with 50 microCi L-[4,5-3H]leucine. Oviductal secretion of de novo synthesized protein measured by incorporation of [3H]leucine into nondialyzable radioactivity in culture supernatants was greatest at D 0 and declined thereafter. Incorporation of [3H]leucine into TCA-precipitable macromolecules in tissue homogenates was also greatest at D 0. Analysis of culture supernatants by one-dimensional polyacrylamide gel electrophoresis revealed four major bands of radiolabeled proteins at greater than 97 kDa, 85-97 kDa, 55 kDa, and 30 kDa. Analysis of individual polypeptides resolved by two-dimensional polyacrylamide gel electrophoresis indicated that five of 32 individual polypeptides examined were secreted at significantly greater rates at estrus than at other times examined. One of these five polypeptides, a 97-kDa peptide with an apparent pI of 5.0, was the major secretory product at estrus and accounted for 18% of total radioactivity recovered from two-dimensional gels. Two of 32 polypeptides examined were secreted at significantly greater rates by explants of the oviduct contralateral to the side of ovulation. In summary, estrus is associated with an elevation in total protein secretion by the bovine oviduct. This increase is due to selective amplification of secretion of several but not all secretory proteins.     

Inhibition of epidermal growth factor-induced phosphorylation by trifluoperazine

We studied the effects of a series of drugs on A431, a cell line with well-characterized growth factor requirements and epidermal growth factor (EGF) receptors. The major [32PO4]-labeled protein immunoprecipitated with anti-phosphotyrosine antibodies from EGF-stimulated A431 cells was the EGF receptor. Both the quantity of [32PO4]-labeled EGF receptor immunoprecipitated and the phosphotyrosine content of total [32PO4]-labeled proteins were reduced by the addition during EGF stimulation of trifluoperazine (TFP). TFP had little effect on the binding, internalization, and processing of [125I]-EGF. In addition to the effects on phosphorylation, TFP inhibited cell growth both in the presence and absence of EGF. Morphologically, TFP blocked EGF-induced ruffling. TFP did not alter the EGF-stimulated phosphatidylinositol turnover. In an in vitro experiment using A431 cell membranes, TFP did not inhibit phosphorylation of the EGF receptor.     

ATP binding properties of the nucleotide-binding folds of SUR1

Pancreatic beta cell ATP-sensitive potassium (K(ATP)) channels regulate glucose-induced insulin secretion. The activity of the K(ATP) channel, composed of SUR1 and Kir6.2 subunits, is regulated by intracellular ATP and ADP, but the molecular mechanism is not clear. To distinguish the ATP binding properties of the two nucleotide-binding folds (NBFs) of SUR1, we prepared antibodies against NBF1 and NBF2, and the tryptic fragment of SUR1 was immunoprecipitated after photoaffinity labeling with 8-azido-[(32)P]ATP. The 35-kDa fragment was strongly labeled with 5 microM 8-azido-[(32)P]ATP even in the absence of Mg(2+) and was immunoprecipitated with the antibody against NBF1. The 65-kDa fragment labeled with 100 microM 8-azido-[alpha-(32)P]ATP in the presence of Mg(2+) was immunoprecipitated with anti-NBF2 and anti-C terminus antibodies. These results indicate that NBF1 of SUR1 binds 8-azido-ATP strongly in a magnesium-independent manner and that NBF2 binds 8-azido-ATP weakly in a magnesium-dependent manner. Furthermore, the 65-kDa tryptic fragment was not photoaffinity-labeled with 8-azido-[gamma-(32)P]ATP at 37 degrees C, whereas the 35-kDa tryptic fragment was, suggesting that NBF2 of SUR1 may have ATPase activity and that NBF1 has none or little.     

Hexose metabolism in pancreatic islets: preferential utilization of mitochondrial ATP for glucose phosphorylation

The respective contribution of exogenous and intramitochondrially formed ATP to D-glucose phosphorylation by mitochondria-bound hexokinase was examined in both rat liver and pancreatic islet mitochondria by comparing the generation of D-glucose 6-[32P]phosphate from exogenous [gamma-32P]ATP to the total rate of D-[U-14C]glucose phosphorylation. In liver mitochondria, the fractional contribution of exogenous ATP to D-glucose phosphorylation ranged from 4 to 74%, depending on the availability of endogenous ATP formed by either oxidative phosphorylation or in the reaction catalyzed by adenylate kinase. Likewise, in islet mitochondria exposed to exogenous ATP but deprived of exogenous nutrient, about 60% of D-glucose phosphorylation was supported by mitochondrial ATP. Such a fractional contribution was further increased in the presence of ADP and succinate, and suppressed by mitochondrial poisons. It is concluded that, in islet like in liver mitochondria, mitochondrial ATP is used preferentially to exogenous ATP as a substrate for D-glucose phosphorylation by mitochondria-bound hexokinase. This may favour the maintenance of a high cytosolic ATP concentration in glucose-stimulated islet cells.     

Change of hepatitis B virions (Dane particles) phosphorylation pattern by human hepatoma cell particulate fraction

Protein kinase activity has been found in hepatitis B virions (Dane particles) purified from the plasma of hepatitis B surface antigen carriers [Albin, C., and Robinson, W.S. (1980) J. Virol. 34, 297-302]. Dane particles were purified from the pooled, HBeAg-positive plasma. When this preparation was incubated with [gamma 32P]ATP in the presence of 10mM MnCl2 and 0.5% NP-40 for 15 seconds at 30 degrees C, several phosphorylated polypeptides of 20,000, 42,000, 48,000, 50,000 and 56,000 daltons were detected in sodium dodecyl sulfate-polyacrylamide gels. When the Dane particles were incubated with [gamma 32P]ATP, 10 mM MnCl2, and 0.5% NP-40 in the presence of human hepatoma cell (J-5) particulate fraction at 30 degrees C, 15 seconds, the 42,000, 48,000 and 50,000 daltons phosphorylated polypeptides were not found. When human peripheral blood lymphocytes particulate fraction was incubated with Dane particles under the same conditions, no change of Dane particle phosphorylated polypeptides was detected. Previous publications [Albin, C., and Robinson, W.S. (1980) J. Virol. 34, 297-302; Gerlich, W.H. et al. (1982) J. Virol. 42, 761-766] showed that when hepatitis B core particles purified from hepatoma tissues contained protein kinase activity, only phosphorylated polypeptide was 20,000 daltons. Our data suggested that when Dane particles were put in an environment of hepatoma cells (or tissues), the protein kinase could only phosphorylate selected polypeptides in these particles.     

Protein phosphorylation in intact bovine epididymal spermatozoa: identification of the type II regulatory subunit of cyclic adenosine 3',5'-monophosphate-dependent protein kinase as an endogenous phosphoprotein

An obstacle to the study of protein phosphorylation in mammalian spermatozoa has been the inability to incorporate sufficient amounts of 32Pi into cellular adenosine triphosphate (ATP) (Babcock et al., 1975). We report conditions under which 32Pi is effectively incorporated into the ATP of intact bovine spermatozoa. In the presence of a bicarbonate-buffered medium containing glucose, spermatozoa incorporated 32P into intracellular ATP in a time-dependent manner; after 2 h of incubation, the specific activity of [gamma-32P]ATP (2.3 X 10(4) cpm/nmol ATP) was estimated to be 50-65% of the specific activity of the intracellular phosphate pool. In the absence of glucose or other added substrates, the specific activity of [gamma-32P]ATP was 10-25% that of the specific activity observed in the presence of glucose. Washed spermatozoa incubated in carrier-free 32Pi for 2 h at 37 degrees C, and solubilized in a solution containing final concentrations of 6.8 M urea, 6% NP4O, and 5% beta-mercaptoethanol contained in excess of 40 32Pi-labeled proteins as assessed by two-dimensional polyacrylamide gel electrophoresis. Major phosphoproteins had approximate molecular weights of 93,000, 40,000, and 22,000. A different two-dimensional gel pattern was observed when cells were extracted with a solution containing 38.5 mM 2[N-cyclohexylamino] ethanesulfonic acid (CHES), pH 9.5/1.5% sodium dodecyl sulphate (SDS) at 100 degrees C. In contrast to the urea/Nonidet P-40 (NP40)/beta-mercaptoethanol extract, a 56,000 Mr phosphoprotein represented a major component while the 40,000 Mr and several of the 22,000 Mr polypeptides were markedly reduced in radioactive intensity. The 56,000 Mr species present in the CHES/SDS extract comigrated with the purified, phosphorylated regulatory subunit (RII) of cyclic adenosine 3',5'-monophosphate-dependent protein kinase from bovine heart. Antibodies to RII immunoprecipitated a 56,000 Mr, 32P-labeled polypeptide from the CHES/SDS extract that comigrated with purified, [32P] RII after two-dimensional electrophoresis. RII, then, appears to represent one of the endogenous phosphoproteins of intact bovine epididymal spermatozoa.     

Regulation of accumulation of the major thylakoid polypeptides in Chlamydomonas reinhardtii y-1 at 25 degrees C and 38 degrees C

The amount of messenger RNA (mRNA) for polypeptides of the chlorophyll a/b-protein complex of thylakoid membranes in etiolated and greening cells of Chlamydomonas reinhardtii y-1 was examined by immunoprecipitation and electrophoresis of products of in vitro translation to determine at which stage production of these polypeptides is regulated. Cells grown 4 d in the dark at 25 degrees C contained small amounts of translatable mRNA for the major membrane polypeptides. Exposure of these etiolated cells to light, under conditions in which the membrane polypeptides accumulated, resulted in a significant increase in the quantity of the mRNA. In contrast, when etiolated cells were incubated for 1-2 h in the dark at 38 degrees C, translation assays indicated that mRNA for the membrane polypeptides became abundant. Moreover, the quantity of the mRNA did not increase when these cells subsequently were exposed to light. Therefore, at 38 degrees C the cellular level of the polypeptides is not regulated by synthesis of mRNA. The in vitro synthesized polypeptides, which were precipitated with antibodies prepared against the purified thylakoid polypeptides, had apparent molecular weights of 31,500 and 30,000. The corresponding immunoprecipitated polypeptides made in vivo had apparent molecular weights of 29,500 and 26,000. Thus, the membrane polypeptides are made as precursors. No net accumulation of the polypeptides occurred in cells in the dark at 38 degrees C, but immunoreactive polypeptides the size of the mature membrane components were labeled during incubation of cells with [14C]acetate in the dark. These results indicated that the mRNA was translated in the dark, but since the polypeptides did not accumulate, the products of translation were probably degraded. We conclude from our experiments that at 25 degrees C production of the polypeptides is regulated by the level of translatable mRNA in the cells. At 38 degrees C, however, the accumulation of the polypeptides is controlled by posttranslational processes.     

Phosphorylation of ATPase subunits of the 26S proteasome

The 26S proteasome complex plays a major role in the non-lysosomal degradation of intracellular proteins. Purified 26S proteasomes give a pattern of more than 40 spots on 2D-PAGE gels. The positions of subunits have been identified by mass spectrometry of tryptic peptides and by immunoblotting with subunit-specific antipeptide antibodies. Two-dimensional polyacrylamide gel electrophoresis of proteasomes immunoprecipitated from [³²P]phosphate-labelled human embryo lung L-132 cells revealed the presence of at least three major phosphorylated polypeptides among the regulatory subunits as well as the C8 and C9 components of the core 20S proteasome. Comparison with the positions of the regulatory polypeptides revealed a minor phosphorylated form to be S7 (MSS1). Antibodies against S4, S6 (TBP7) and S12 (MOV34) all cross-reacted at the position of major phosphorylated polypeptides suggesting that several of the ATPase subunits may be phosphorylated. The phosphorylation of S4 was confirmed by double immunoprecipitation experiments in which 26S proteasomes were immunoprecipitated as above and dissociated and then S4 was immunoprecipitated with subunit-specific antibodies. Antibodies against the non-ATPase subunit S10, which has been suggested by others to be phosphorylated, did not coincide with the position of a phosphorylated polypeptide. Some differences were observed in the 2D-PAGE pattern of proteasomes immunoprecipitated from cultured cells compared to purified rat liver 26S proteasomes suggesting possible differences in subunit compositions of 26S proteasomes.     

Remodeling of a rat hepatocyte plasma membrane glycoprotein. De- and reglycosylation of dipeptidyl peptidase IV

The present paper demonstrates the terminal de- and reglycosylation of a rat hepatocyte plasma membrane glycoprotein, dipeptidyl peptidase IV (DPP IV). Cultured hepatocytes were used in pulse-chase experiments with [3H]L-fucose and [14C]N-acetyl-D-mannosamine as markers for terminal carbohydrates, [3H]D-mannose as marker of a core-sugar, and [35S]L-methionine for labeling the protein backbone. Membrane DPP IV was immunoprecipitated with a polyclonal antibody which bound selectively at 4 degrees C to the cell-surface glycoprotein. The times of maximal labeling of hepatocyte plasma membrane DPP IV were 6-9 min for [3H]L-fucose, 20 min for [3H]D-mannose, and 25 min for [35S]L-methionine. When antibodies were bound to cell-surface DPP IV at 4 degrees C, the immune complex remained stable for more than 1 h after rewarming to 37 degrees C, despite ongoing metabolic and membrane transport processes. This was shown by pulse labeling with [35S]L-methionine at 37 degrees C, followed by cooling to 4 degrees C, and addition of antibody against plasma membrane DPP IV. During rewarming, the radioactivity in the complex remained constant. In a similar experiment with [3H]L-fucose, the radioactivity in the immune complex declined rapidly, indicating a defucosylation of the plasma membrane glycoprotein. Using the same experimental design with [3H]D-mannose, the radioactivity in the immune complex remained constant, showing that the core-sugar D-mannose is not cleaved from the membrane glycoprotein. Terminal reglycosylation (refucosylation and resialylation) was demonstrated as follows. Hepatocytes were maintained at 37 degrees C in a medium supplemented with tunicamycin in order to block the de novo synthesis of N-glycosidically bound carbohydrate chains. At 4 degrees C the antibody against DPP IV bound only to cell surface glycoprotein. During the rewarming period at 37 degrees C, radioactivity from [3H]L-fucose and [14C]N-acetyl-D-mannosamine became incorporated into the immune complex. This indicates a fucosylation and sialylation of the glycoprotein originally present at the cell surface. The mechanisms whereby terminal de- and reglycosylation of plasma membrane glycoproteins may occur during membrane recycling are discussed.     

Development and application of a system for seminolipid metabolism using mouse seminiferous tubules

A convenient tool for studying metabolism of seminolipid in testis was developed by using mouse isolated seminiferous tubules prepared by collagenase treatment. Because more than 99% of [³⁵S]sulfate-incorporation was distributed in seminolipid, its metabolism in seminiferous tubules can be analyzed without disturbance of the other sulfolipids in this assay system. Furthermore, the contents of seminolipid and its precursor, galactosylalkylacylglycerol, which were determined by liquid chromatography-electrospray ionization mass spectrometry, did not change within a few hours, indicating that the incorporations of [³⁵S]sulfate into seminolipid solely reflects the turnover rate of this sulfolipid. As an initial application of this system, we characterized heat-susceptibility of the seminolipid turnover rate in mouse seminiferous tubules. Severe heating (44°C for 10 min) of the isolated seminiferous tubules suppressed the ³⁵S-incorporation into seminolipid to 47% of heating at scrotal temperature (32°C for 70 min). In contrast, pretreatment of the testis in vivo under the same condition (44°C for 10 min) did not decrease the seminolipid turnover rate in the isolated seminiferous tubules. In addition, the activity of galactocerebroside sulfotransferase decreased in the temperature-dependent manner in seminiferous tubules as well as crude tubular homogenates, where the activity is significantly more stable in the former than the latter. The newly developed system could provide useful basic data for further analyses of seminolipid metabolism in the testis.     

Polypeptide components of the apamin receptor associated with a calcium activated potassium channel

Photoaffinity labeling of rat brain membranes with [125I]ANPAA-apamin incorporated radioactivity into polypeptides of 86 and 59 kDa and occasionally a more weakly labeled component of 45 kDa. These polypeptides were immunoprecipitated with anti-apamin antibodies and treated with glycosidases. Neither the 86 nor the 59 kDa polypeptide appeared to be N-glycosylated. Partial proteolytic mapping of affinity labeled polypeptides with chymotrypsin or V8 protease generated an identical pattern. These results suggest that the 59 and 45 kDa components are not additional subunits of an oligomeric protein but result from cleavage of the 86 kDa polypeptide.     

Subunit composition and ATP site labeling of the coated vesicle proton-translocating adenosinetriphosphatase

The partially purified proton-translocating adenosinetriphosphatase [(H+)-ATPase] from clathrin-coated vesicles has been reported to contain eight polypeptides of molecular weights 15,000-116,000 [Xie, X.S., & Stone, D.K. (1986) J. Biol. Chem. 261, 2492-2495]. To determine whether these polypeptides form a single macromolecular complex, we have isolated three monoclonal antibodies which recognize the reconstitutively active (H+)-ATPase in the native, detergent-solubilized state. All three monoclonal antibodies precipitate the same set of polypeptides from either the partially purified enzyme or the detergent-solubilized coated vesicle membrane proteins. The immunoprecipitated polypeptides have molecular weights of 100,000, 73,000, 58,000, 40,000, 38,000, 34,000, 33,000, 19,000, and 17,000. These results thus indicate that this set of polypeptides forms a single macromolecular complex and suggest that they correspond to subunits of the coated vesicle (H+)-ATPase. To identify the ATP-hydrolytic subunit of the coated vesicle (H+)-ATPase, the purified enzyme was reacted with N-ethylmaleimide (NEM) and 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl), both of which inhibit activity in an ATP-protectable manner. Labeling was carried out by using [3H]NEM or [14C]NBD-Cl, and the specificity of the reaction was increased by prelabeling of the protein with the nonradioactive reagents in the presence of ATP and by taking advantage of the nucleotide specificity of protection. The principal polypeptide labeled by both [3H]NEM and [14C]NBD-Cl had a molecular weight of 73,000. In addition, this protein was the only polypeptide whose labeling was significantly reduced in the presence of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoradiographic study of thermo-dependent nucleo-cytoplasmic distribution of aldosterone binding sites in intact target cells

Autoradiographic studies of [3H]aldosterone [( 3H-A] and [3H]dexamethasone binding sites in intact target cells (isolated collecting tubules of rabbit and rat kidney) revealed an almost exclusive nuclear localization of the hormone-receptor complexes. In the present work we compared the nucleo-cytoplasmic repartition of [3H]A-receptor complexes studied in parallel by biochemical and autoradiographic methods. In addition, the thermo-dependency of the nuclear translocation was examined. Kidney pyramids were incubated in vitro with [3H]A (2 X 10(-9) M) in the presence or absence of a 100-fold excess unlabelled A, at 30 degrees C for 1 h or 4 degrees C for 2 h. Then tissue was processed for isolation of nuclear and cytoplasmic fractions, on the one hand, or for obtention of microdissected tubular segments on which autoradiographs on dry films were performed. Autoradiographs showed that the specific labelling was almost exclusively nuclear without significant cytoplasmic labelling, at both 30 or 4 degrees C. This indicates that almost all binding sites migrated rapidly into nuclei, and that this translocation did not depend on temperature. In contrast, parallel biochemical experiments yielded classical results, that is, at 30 degrees C, the presence of specific binding sites in both cytoplasm and nuclei with a predominance in cytoplasm. At 4 degrees C, the cytoplasmic binding was unchanged, but nuclear binding was drastically reduced, indicating thermodependency of nuclear translocation, when studied by biochemical methods including cell disruption. Autoradiographic results thus questioned the classical notion of thermo-dependent nuclear translocation of aldosterone-receptor complexes, based on results obtained by biochemical methods.     

Interconversion of D-fructose 1,6-bisphosphate and triose phosphates in human erythrocytes.

Aldolase and triose phosphate isomerase both display strict specificity towards the enantiomers of [1-3H]glycerone 3-phosphate. The enantiomer generated from D-[1-3H]glyceraldehyde 3-phosphate produces 3HOH in the aldolase reaction, whilst the other enantiomer generated from D-[3-3H]fructose 1,6-bisphosphate is solely detritiated in the reaction catalyzed by triose phosphate isomerase. Advantage was taken of such a specificity to assess, in human erythrocytes exposed to either D-[3-3H]glucose or D-[3,4-3H]glucose, the extent of D-glyceraldehyde 3-phosphate sequential conversion to glycerone 3-phosphate and D-fructose 1,6-bisphosphate, relative to net glycolytic flux. At 37 degrees C and in the presence of 5.6 mM D-glucose, only 55% of the metabolites of D-[4-3H]glucose underwent detritiation in the reactions catalyzed by triose phosphate isomerase and aldolase. Such a percentage was further decreased at low temperature (8 degrees C) or lower concentrations of D-glucose (0.2 and 1.0 mM). However, when the erythrocytes were exposed to menadione, the increase in 3HOH production from either D-[3-3H]glucose or D-[3,4-3H]glucose indicated that the majority of the 3H atoms initially located on the C4 of D-glucose were recovered as 3HOH upon circulation through the pentose phosphate pathway. These findings suggest that, under physiological conditions, a large fraction of D-glyceraldehyde 3-phosphate generated from exogenous D-glucose may undergo enzyme-to-enzyme channelling in the glycolytic pathway.     

Effects of temperature on the degradation and biliary secretion of asialoorosomucoid by the perfused rat liver: evidence for two intracellular pathways

We have utilized the in situ perfused rat liver under nonrecirculating conditions to examine the effect of temperature on the metabolism and biliary secretion of [125I]-asialoorosomucid (ASOR). In this manner we were able to follow the fate of a single round of internalized ligand. In control livers perfused at 37 degrees C, approximately 50% of [125I]-ASOR injected into the portal vein was extracted on first pass. Five minutes after the injection, radioactivity, which had been extracted initially, began to appear in the hepatic venous effluent. Within 25 min, 50% of the initially extracted radioactivity was released into the perfusion medium; the bulk of this radioactivity (greater than 95%) was soluble in trichloroacetic acid. In livers perfused at temperatures slightly less than 37 degrees C (30-35 degrees C), first-pass extraction of [125I]-ASOR was similar to that observed at 37 degrees C. However, a severalfold decrease in the rate of release of radioactivity from the liver into the perfusion medium was noted at the lower perfusion temperatures; whereas greater than 50% of the initially extracted radioactivity was released within 30 min from livers perfused at 37 degrees C, only 5% was released at 30 degrees C. At the lower perfusion temperature, a larger proportion of the released radioactivity was acid precipitable (24% vs. 5%). Some radioactivity also was recovered in the bile; of the total amount of radioactivity released from the liver in 30 min at 37 degrees C, approximately 5% was directed into the bile. At lower temperatures of perfusion, a greater fraction of the radioactivity that was released from the liver was directed into the bile (20% at 30 degrees C vs. 5% at 37 degrees C). The data imply that the endosomal pathway to the lysosome is highly sensitive to slight reductions in temperature while the transcytotic route into bile is less sensitive. Lower temperatures might prolong the residence time of ASOR in the prelysosomal endosomal compartments, and thereby increase the likelihood that undegraded ligand will be returned to the blood or be missorted into bile.     

Effects of vasectomy on the blood-testis barrier of the hamster.

The effects of vasectomy on the blood-testis and blood-epididymal barriers to 3H2O, [3H]inulin, and [14C]urea were examined by study of the radioactivity appearing in micropuncture samples of fluids from the seminiferous tubules and cauda epididymidis. By 4 months after vasectomy, there were changes in the blood-seminiferous tubule barrier to [3H]water and [14C]urea (increased entry) and in the blood-epididymal barrier to [3H]water and [3H]inulin (increased entry) and to [14C]urea (decreased entry). These subtle changes could have an impact on spermatogenesis and/or sperm maturation after vasectomy.     

Anomeric specificity of D-[U-14C]glucose incorporation into glycogen in rat hemidiaphragms

The anomeric specificity of D-[U-14C]glucose incorporation into glycogen in rat hemidiaphragms was investigated. For this purpose, the hemidiaphragms were preincubated for 30 min at 37 degrees C and then incubated for 5 min at the same temperature in the presence of alpha- or beta-D-[U-14C]glucose. The concentrations of D-glucose (5.6 or 8.8 mM) and insulin (0 or 10 mU/ml) were identical during the preincubation and incubation periods. The incubation medium was prepared in D2O/H2O (3:1, v/v) in order to delay the interconversion of the D-glucose anomers. In addition to glycogen labelling, the output of radioactive acidic metabolites was also measured. Insulin caused a preferential stimulation of glycogen labelling relative to glycolysis. Such was not the case in response to a rise in D-glucose concentration. At 5.6 mM D-glucose and whether in the presence or absence of insulin, both glycogen labelling and glycolysis were lower with alpha-D-glucose than with beta-D-glucose suggesting a higher rate of beta-D-glucose than alpha-D-glucose transport across the plasma membrane. A mirror image was found at 8.8 mM D-glucose, especially in the absence of insulin. At this close-to-physiological hexose concentration, insulin lowered the alpha/beta ratio for glycogen labelling. On the contrary, the rise in D-glucose concentration increased such a ratio. Since such a rise is probably little affected by any possible anomeric difference in D-glucose transport across the plasma membrane, the present results strongly suggest that the intracellular factors regulating net glycogen synthesis, as well as glycolytic flux, display obvious preference for alpha-D-glucose.     

The effect of cycloheximide on the glycosylation of lactating-rabbit mammary glycoproteins.

1. Cycloheximide inhibited immediately the incorporation of L-[4,5-3H]leucine and D-]2-3H]mannose into mammary proteins, suggesting that the mannosylation of mammary glycoproteins requires the continued supply of newly synthesized polypeptides. 2. The incorporation of radioactivity from N-acetyl-D-[1-14C] glucosamine into protein was not inhibited until approx. 30 min after cycloheximide addition. Much (greater than 90%) of this radioactivity was present as N-acetylgalactosamine. 3. N-Glycosylation appears to be inhibited immediately by cycloheximide due to a lack of newly synthesized acceptor polypeptides, whereas O-glycosylation continues for 30 min, the time taken for acceptor peptides to move from their site of synthesis to the Golgi region and for completion of glycosylation. 4. There was a transient increase in the incorporation of mannose into lipid-linked oligosaccharide in the presence of cycloheximide, followed by a decrease in the radioactivity in this fraction. 5. The major lipid-linked oligosaccharide extracted from explants incubated for 2h in the presence of cycloheximide (6-7 monosaccharide units) was smaller than that extracted from control explants (10-12 monosaccharide units).     

Comparative analysis of proteins from the fibrous sheath and outer dense fibers of rat spermatozoa

The protein composition of the fibrous sheath (FS) and the outer dense fibers (ODF), two cytoskeletal components of the tail of spermatozoa, was compared by using polyacrylamide gel electrophoresis and immunochemistry applied to Western blots and to spermatozoa. Isolated FS and ODF, the purity of which were verified by electron microscopy (EM), were denatured and either run on sodium dodecyl sulfate-polyacrylamide gels or used to raise antibodies. The gels revealed at least 18 and 14 polypeptide bands for the FS and ODF, respectively. The major bands of the FS had molecular masses of 75, 27.5, and 14.4 kDa, whereas the major bands of the ODF-connecting piece had molecular masses of 32-26, 20, 14.4, 84, and 80 kDa. Several prominent FS and ODF bands were found to comigrate on gels, and the 14.4 kDa polypeptides had similar electrophoretic properties. Anti-FS serum reacted with the majority of Western blot-transferred FS polypeptides, but also cross-reacted strongly with a major 14.4 kDa ODF polypeptide and with less affinity to other major ODF polypeptides. Anti-ODF serum reacted with the majority of ODF polypeptides, but also cross-reacted strongly with a major 14.4 kDa FS polypeptide, and with less affinity to several other FS polypeptides including the 75 kDa band. Antibodies affinity-purified from the 14.4 kDa FS polypeptide only cross-reacted with the 14.4 kDa ODF polypeptide, whereas antibodies purified from the 14.4 kDa ODF polypeptide cross-reacted with 14.4, 27.5, 57, and 63 kDa FS polypeptides. The immunocross-reactions observed on Western blots were confirmed by immunocytochemical methods applied to spermatozoa. This study demonstrates that the FS and ODF, both composed of many polypeptides, several having similar molecular weights, are related cytoskeletal structures as they have epitopes in common, and both contain 14.4 kDa polypeptides with common antigenic and electrophoretic properties.     

Synthesis and degradation of unassembled polypeptides of the coupling factor of photophosphorylation CF1 in 70S ribosome-deficient rye leaves

The formation of polypeptides of the coupling factor CF1 was investigated in 70S ribosome-deficient rye leaves generated by growing the plants at a non-permissive elevated temperature of 32 degrees C, in order to analyse mechanisms coordinating subunit accumulation. Antibodies were raised in rabbits against total CF1 as well as against its five individual subunits purified from chloroplast thylakoids from rye leaves. Several immunological techniques applying these antibodies (immunoprecipitation, immunoblotting, antibody affinity chromatography) were unable to detect the presence of any of the CF1 subunits in heat-treated 70S ribosome-deficient leaves. After in vivo labeling with L-[35S]methionine and subsequent immunoprecipitation, however, radioactivity was found to be incorporated into the subunits gamma and delta, but not into alpha, beta and epsilon, in 70S ribosome-deficient leaves, demonstrating the cytoplasmic synthesis of CF1-gamma and CF1-delta. Chase experiments after in vivo labeling with L-[35S]methionine indicated that the unassembled subunits gamma and delta were rapidly and preferentially degraded, while they were stabilized when integrated into the complete CF1 complex in normal green leaves from permissive growth conditions. The apparent half-times of the unassembled subunits were 2 h for CF1-gamma and 4 h for CF1-delta in 32 degrees C-grown leaves. Several other, stromal, plastid proteins of cytoplasmic origin were stable in 32 degrees C-grown leaves during the period of chase. In etiolated leaves total CF1, including all subunits, appeared to be less stable than in green leaves grown under permissive temperature conditions in light. Rapid degradation of the excess of unassembled subunits is regarded as an important mechanism ensuring a constant stoichiometry and apparently synchronous development of CF1 subunits.