Physicochemical properties and specific structural features of protein-lipid composites of increased nutritive value

Fractional and component compositions of protein-lipid composites with increased nutritive value (compared to the protein preparations from which they were produced) were studied based on solubility and electrophoretic behavior. Differences in the fractional compositions of proteins and the amounts of hydrogen, ionic, and hydrophobic bonds were found. It was demonstrated that the water-, salt-, and alkali-soluble fractions of proteins changed during the manufacturing of the composites with soybean and wheat bran flour; the water- and alkali-soluble fractions, with protein concentrate from bran. Heterogeneity of the compositions and specific conformational features of composite proteins resulting from disulfide bonds were found. It was demonstrated that, during the manufacturing of composites, proteins of soybean flour aggregated (with the involvement of disulfide bonds), whereas protein products from wheat bran disaggregated. Breaks of interchain (wheat) or intrachain (concentrate) disulphide bonds accompanied the disaggregation. Overall the properties and specific structural features of the protein-lipid composites studied depended on the nature of the protein (soybean or wheat), type of initial preparations (flour or concentrate), and method of their production (emulsifying or drying).     

Physicochemical Properties and Specific Structural Features of Protein–Lipid Composites of Increased Nutritive Value

Fractional and component compositions of protein–lipid composites with increased nutritive value (compared to the protein preparations from which they were produced) were studied based on solubility and electrophoretic behavior. Differences in the fractional compositions of proteins and the amounts of hydrogen, ionic, and hydrophobic bonds were found. It was demonstrated that the water-, salt-, and alkali-soluble fractions of proteins changed during the manufacturing of the composites with soybean and wheat bran flour; the water- and alkali-soluble fractions, with protein concentrate from bran. Heterogeneity of the compositions and specific conformational features of composite proteins resulting from disulfide bonds were found. It was demonstrated that, during the manufacturing of composites, proteins of soybean flour aggregated (with the involvement of disulfide bonds), whereas protein products from wheat bran disaggregated. Breaks of interchain (wheat) or intrachain (concentrate) disulphide bonds accompanied the disaggregation. Overall the properties and specific structural features of the protein–lipid composites studied depended on the nature of the protein (soybean or wheat), type of initial preparations (flour or concentrate), and method of their production (emulsifying or drying).     

Changes in the Activity of the Maturation-Promoting Factor Are Correlated with Those of a Major Cyclic AMP and Calcium-Independent Protein Kinase During the First Mitotic Cell Cycles in the Early Starfish Embryo

Many studies suggest that MPF activation depends on protein phosphorylation or that MPF is itself a protein kinase. In the present report, cyclic variations of MPF activity have been correlated in vivo with changes in the extent of protein phosphorylation or in vitro with changes of a major protein kinase during the first cell cycles of fertilized starfish eggs. This cycling protein kinase neither requires cAMP nor Ca²⁺. Neither colchicine nor aphidicoline, which inhibits cleavage and chromosome replication respectively, was found to suppress the synchronous and cyclic variations of both MPF and protein kinase activities. Protein synthesis was found to be required for both MPF and protein kinase activities to reappear after their simultaneous drop at the time of mitotic or meiotic cleavages. Production of either MPF or protein kinase activities is not the immediate result of protein synthesis since there is a delay at each cell cycle between the time when protein synthesis is required and the time when both MPF and protein kinase activities are produced. This suggests that both MPF and protein kinase activities might involve some post-translational modification of a precursor protein synthesized during the preceeding cell cycle.     

Peroxynitrite mediates programmed cell death both in papillar cells and in self-incompatible pollen in the olive (Olea europaea L.)

Programmed cell death (PCD) has been found to be induced after pollination both in papillar cells and in self-incompatible pollen in the olive (Olea europaea L.). Reactive oxygen species (ROS) and nitric oxide (NO) are known to be produced in the pistil and pollen during pollination but their contribution to PCD has so far remained elusive. The possible role of ROS and NO was investigated in olive pollen-pistil interaction during free and controlled pollination and it was found that bidirectional interaction appears to exist between the pollen and the stigma, which seems to regulate ROS and NO production. Biochemical evidence strongly suggesting that both O(2)(-) and NO are essential for triggering PCD in self-incompatibility processes was also obtained. It was observed for the first time that peroxynitrite, a powerful oxidizing and nitrating agent generated during a rapid reaction between O(2)(-) and NO, is produced during pollination and that this is related to an increase in protein nitration which, in turn, is strongly associated with PCD. It may be concluded that peroxynitrite mediates PCD during pollen-pistil interaction in Olea europaea L. both in self-incompatible pollen and papillar cells.     

Microarrays and the relationship of mRNA variation to protein variation during the cell cycle

Microarray analyses have led to the postulated existence and identification of numerous genes that are believed to be expressed and presumably to act in a cell-cycle-specific manner because their expression varies during the cell cycle. It is important to see how protein variation can be produced from mRNA variation. We have calculated the protein content throughout the cell cycle resulting from cell-cycle-specific mRNA expression, and compared the result to protein content resulting from constant, cell-cycle independent, mRNA expression. For stable proteins, cell-cycle-specific mRNA expression leads to a maximum 2-fold change in protein content compared to proteins synthesized from constantly expressed mRNA. More realistic sinusoidal patterns of mRNA expression exhibit much smaller ratios of 1.25 or lower, even for extremely large amplitudes in mRNA expression. For unstable proteins that have a cycle-independent half-life, only at extremely short protein half-lives does mRNA variation have a significant impact on variation of protein content during the division cycle. We also apply these findings to proteins with a cycle-specific decay pattern. mRNA variations during the eukaryotic division cycle variation of mRNA during the cell cycle can have only a minimal affect on the variation of protein content during the cell cycle. We conclude that mRNA variations during the division cycle, as measured by microarrays, cannot by themselves, identify cycle-specific functions related to protein variations.     

Variation in the expression of pili and outer membrane protein by Neisseria meningitidis during the course of meningococcal infection

The occurrence of antigenic shift during meningococcal infection has been investigated by comparison of paired isolates obtained from the blood, cerebrospinal fluid or nasopharynx of patients. Isolates from any individual produced identical DNA 'fingerprints' and showed stability in expression of both class 2 outer membrane protein and an antigen common to pathogenic Neisseria, confirming their origin as a single strain. One of the four strains examined produced variants which differed in the molecular mass of their class 5 outer membrane proteins. Three of the strains produced pili containing the epitope recognized by monoclonal antibody SM1 and two of these gave rise to variants which expressed pili of differing subunit molecular masses. The two variants of the remaining strain produced pilins lacking the common epitope detected by antibody SM1 but radioimmune precipitation with polyclonal anti-pilus antiserum revealed that variation in the molecular mass of the pilin expressed also occurred with this second class of pili. Antigenic variation in expression of both class 5 outer membrane proteins and pili therefore appears to be a common occurrence during meningococcal infection.     

The interrelationship of the soluble and membrane-associated folate-binding proteins in human KB cells

Human KB cells produce two immunologically cross-reactive folate-binding proteins: a particulate cell-associated protein which is solubilized by Triton X-100, and a soluble protein which is released into their growth medium. This compartmentation of these two folate-binding proteins provides a convenient system for studies of their biochemical relationship. The two folate-binding proteins behave similarly to the purified particulate and soluble folate-binding proteins of human milk in analysis by radioactive folate binding, Sephacryl S-200 gel filtration profiles, polyacrylamide gel electrophoresis in either Triton X-100 or sodium dodecyl sulfate, and in Triton X-100 binding based on sucrose density gradient ultracentrifugation in H2O and D2O. The two folate-binding proteins were endogenously labeled by pulsing methionine-starved KB cells with [35S]methionine, and each protein was purified to apparent homogeneity by affinity chromatography at different times during the chase with nonradioactive methionine. The time course of the changes in specific activity (moles of [35S]methionine per mole of folate-binding protein) revealed a more rapid initial rate of synthesis and an earlier maximum in specific activity for the cell-associated folate-binding protein than for the soluble folate-binding protein released into the growth medium. Differences in the levels and specific activities of the two folate-binding proteins of cells exposed to cycloheximide compared with simultaneous controls after pulsing with [35S]methionine suggest that, whereas the cell-associated folate-binding protein is probably produced by de novo protein synthesis, the soluble folate-binding protein seems to be produced from a cellular pool of an already synthesized protein. These results combined with the immunologic cross-reactivity of the two folate-binding proteins strongly suggest a precursor-product relationship between them.     

Calpain II induced insolubilization of lens β-crystallin polypeptides may induce cataract

Addition of calpain II (EC 3.4.22.17) to soluble proteins from 10-day-old rat lens caused an increase in turbidity and production of water-insoluble protein. The insolubilization increased with higher concentrations of both lens protein and calpain II, it could be prevented by the cysteine protease inhibitor E-64; it required at least 0.5 mM Ca²⁺, it was limited to 6% of the soluble protein present and resulted from precipitation β-crystallin polypeptides. When compared by two-dimensional electrophoresis, the insoluble β-crystallin polypeptides produced by calpain II were similar to insoluble β-crystallin polypeptides found incataractous lenses. Trypsin also caused insolubilization of β-crystallin polypeptides, but these polypeptides were unlike polypeptides produced during cataract formation. These data suggested that the loss of solubility was due to a specific removal of N/or C-terminal extensions from β-crystallin polypeptides by calpain II, and that a similar process may occur in vivo during cataract formation. It is hypothesized that the insoluble protein produced by calpain II causes cataract by increasing light scatter in the lens.     

Proteome analysis of aerobic and fermentative metabolism in Rhizobium etli CE3

Rhizobium etli undergoes a transition from an aerobic to a fermentative metabolism during successive subcultures in minimal medium. This metabolic transition does not occur in cells subcultured in rich medium, or in minimal medium containing either biotin or thiamine. In this report, we characterize the aerobic and fermentative metabolism of R. etli using proteome analysis. According to their synthesis patterns in response to aerobic (rich medium, minimal medium with biotin or minimal medium with thiamine) or fermentative (minimal medium without supplements) growth conditions, proteins were assigned to five different classes: (i) proteins produced only in aerobic conditions (e.g., catalase-peroxidase KatG and the E2 component of pyruvate dehydrogenase); (ii) protein produced under both conditions but strongly induced in aerobic metabolism (e.g., malate dehydrogenase and the succinyl-CoA synthetase beta subunit); (iii) proteins that were induced equally under all conditions tested (e.g., AniA, DnaK, and GroEL); (iv) proteins downregulated during aerobic metabolism, and (v) proteins specific to only one of the conditions analyzed. Northern blotting studies of katG expression confirmed the proteome data for this protein. The negative regulation of carbon metabolism proteins observed in fermentative metabolism is consistent with the drastic physiological changes which occur during this process.     

In Vitro Generation from the trans-Golgi Network of Coatomer-Coated Vesicles Containing Sialylated Vesicular Stomatitis Virus-G Protein

We describe an in vitro system in which post-Golgi vesicles containing metabolically labeled, sialylated, vesicular stomatitis virus (VSV) G protein molecules (VSV-G) are produced from the trans-Golgi network (TGN) of an isolated Golgi membrane fraction. This fraction is prepared from VSV-infected Madin–Darby canine kidney (MDCK) cells in which the ³⁵S-labeled viral envelope glycoprotein was allowed to accumulate in the trans-Golgi network during a prolonged incubation at 20°C. The vesicles produced in this system are separated from the remnant Golgi membranes by differential centrifugation or by velocity sedimentation in a sucrose gradient. Vesicle production, quantified as the percentage of labeled VSV-G released from the Golgi membranes, is optimal at 37°C and does not occur below 20°C. It requires GTP and the small GTP-binding protein Arf (ADP-ribosylation factor), as well as coat protein type I (COPI) coat components (coatomer) and vesicle scission factors—one of which corresponds to the phosphatidylinositol transfer protein (PITP). Formation of the vesicles does not require GTP hydrolysis which, however, is necessary for their uncoating. Thus, vesicles generated in the presence of the nonhydrolyzable GTP analogs, GTPγS or GMP–PNP, retain a coatomer coat visible in the electron microscope, sediment more rapidly in sucrose density gradients than those generated with ATP or GTP, and can be captured with anticoatomerantibodies. The process of coatomer-coated vesicle formation from the TGN can be dissected into two distinct sequential phases, corresponding to coat assembly/bud formation and vesicle scission. The first phase is completed when Golgi fractions are incubated with cytosolic proteins and nonhydrolyzable GTP analogs at 20°C. The scission phase, which leads to vesicle release, takes place when coated Golgi membranes, recovered after phase I, are incubated at higher temperatures in the presence of cytosolic proteins. The scission phase does not take place if protein kinase C inhibitors are added during the first phase, even though these inhibitors do not prevent membrane coating and bud formation. The phosphorylating activity of a protein kinase C, however, plays no role in vesicle formation, since this process does not require ATP.     

Glycoprotein nature of yeast alkaline phosphatase. Formation of active enzyme in the presence of tunicamycin.

The nonspecific alkaline phosphatase of yeast (Saccharomyces strain 1710) has been purified by ion exchange, hydrophobic, and affinity chromatography. This vacuolar enzyme has a molecular weight of 130,000 and is composed of subunits (probably of 66,000 molecular weight). It also has a small quantity of covalently associated carbohydrate; hydrolysis yielded mannose and glucosamine. The endo-beta-N-acetylglucosaminidase of Streptomyces plicatus released carbohydrate indicating that the latter was attached to protein through an N-acetylglucosaminylasparginyl bond. Synthesis of active alkaline phosphatase by yeast protoplasts is not depressed by tunicamycin, an inhibitor of dolichol-mediated protein glycosylation. Unlike the enzyme normally produced, the alkaline phosphatase which is formed in the presence of the antibiotic does not interact with concanavalin A and, therefore is deficient in or lacking carbohydrate. We infer that there is no regulatory link in yeast between the glycosylation of a protein and its synthesis. The fact that other Asn-GlcNAc-type glycoprotein enzymes of yeast such as acid phosphatase are not produced in their active forms by tunicamycin-treated protoplasts may mean that, as unglycosylated proteins, they cannot be correctly folded or processed. Protoplasts derepressed for phosphatase production contained substantial amounts of a second alkaline phosphatase which differed from the purified enzyme in substrate specificity, sensitivity to calcium, and reactivity with concanavalin A.     

Peroxynitrite inhibits T lymphocyte activation and proliferation by promoting impairment of tyrosine phosphorylation and peroxynitrite-driven apoptotic death

Peroxynitrite (ONOO-) is a potent oxidizing and nitrating agent produced by the reaction of nitric oxide with superoxide. It readily nitrates phenolic compounds such as tyrosine residues in proteins, and it has been demonstrated that nitration of tyrosine residues in proteins inhibits their phosphorylation. During immune responses, tyrosine phosphorylation of key substrates by protein tyrosine kinases is the earliest of the intracellular signaling pathways following activation through the TCR complex. This work was aimed to evaluate the effects of ONOO- on lymphocyte tyrosine phosphorylation, proliferation, and survival. Additionally, we studied the generation of nitrating species in vivo and in vitro during immune activation. Our results demonstrate that ONOO-, through nitration of tyrosine residues, is able to inhibit activation-induced protein tyrosine phosphorylation in purified lymphocytes and prime them to undergo apoptotic cell death after PHA- or CD3-mediated activation but not upon phorbol ester-mediated stimulation. We also provide evidence indicating that peroxynitrite is produced during in vitro immune activation, mainly by cells of the monocyte/macrophage lineage. Furthermore, immunohistochemical studies demonstrate the in vivo generation of nitrating species in human lymph nodes undergoing mild to strong immune activation. Our results point to a physiological role for ONOO- as a down-modulator of immune responses and also as key mediator in cellular and tissue injury associated with chronic activation of the immune system.     

Catalysis of ACAT may be completed within the plane of the membrane: a working hypothesis.

Two ACAT sharing protein sequence homology near their C termini have been identified. Both proteins may span the endoplasmic reticulum (ER) membrane several times. There is good evidence implicating the role of ACAT1 in macrophage foam cell formation, and ACAT2 in intestinal cholesterol absorption. On the other hand, the functional roles of ACAT1 and ACAT2 in the VLDL or chylomicron assembly process are less clear. It is possible that both enzymes are able to form lipid droplets (which are present in the cytoplasm), and participate in lipoprotein assembly (which occurs in the ER lumen). To link the site of ACAT catalysis with its function, we propose that part of the ACAT catalytic site may reside within the lipid bilayer, allowing catalysis to be completed within the plane of the membrane. Cholesteryl esters (CE) produced in situ may burst into cytoplasmic lipid droplets, carrying phospholipid monolayers as their outer coats. In cells engaged in lipoprotein assembly and secretion, CE in the bilayer may be recognized by the specific protein microsomal triacylglycerol transfer protein (MTP), reaching out from the lumenal side of the membrane. MTP then lipidates the growing apolipoprotein B (apoB) chain with CE and TG during the early stages of apoB lipoprotein assembly.     

In vitro generation from the trans-Golgi network of coatomer-coated vesicles containing sialylated vesicular stomatitis virus-G protein

We describe an in vitro system in which post-Golgi vesicles containing metabolically labeled, sialylated, vesicular stomatitis virus (VSV) G protein molecules (VSV-G) are produced from the trans-Golgi network (TGN) of an isolated Golgi membrane fraction. This fraction is prepared from VSV-infected Madin-Darby canine kidney (MDCK) cells in which the (35)S-labeled viral envelope glycoprotein was allowed to accumulate in the trans-Golgi network during a prolonged incubation at 20 degrees C. The vesicles produced in this system are separated from the remnant Golgi membranes by differential centrifugation or by velocity sedimentation in a sucrose gradient. Vesicle production, quantified as the percentage of labeled VSV-G released from the Golgi membranes, is optimal at 37 degrees C and does not occur below 20 degrees C. It requires GTP and the small GTP-binding protein Arf (ADP-ribosylation factor), as well as coat protein type I (COPI) coat components (coatomer) and vesicle scission factors-one of which corresponds to the phosphatidylinositol transfer protein (PITP). Formation of the vesicles does not require GTP hydrolysis which, however, is necessary for their uncoating. Thus, vesicles generated in the presence of the nonhydrolyzable GTP analogs, GTPgammaS or GMP-PNP, retain a coatomer coat visible in the electron microscope, sediment more rapidly in sucrose density gradients than those generated with ATP or GTP, and can be captured with anticoatomerantibodies. The process of coatomer-coated vesicle formation from the TGN can be dissected into two distinct sequential phases, corresponding to coat assembly/bud formation and vesicle scission. The first phase is completed when Golgi fractions are incubated with cytosolic proteins and nonhydrolyzable GTP analogs at 20 degrees C. The scission phase, which leads to vesicle release, takes place when coated Golgi membranes, recovered after phase I, are incubated at higher temperatures in the presence of cytosolic proteins. The scission phase does not take place if protein kinase C inhibitors are added during the first phase, even though these inhibitors do not prevent membrane coating and bud formation. The phosphorylating activity of a protein kinase C, however, plays no role in vesicle formation, since this process does not require ATP.     

High level expression of grass carp reovirus VP7 protein in prokaryotic cells

Sequences analysis revealed Grass carp reovirus (GCRV) s10 was 909 nucleotides coding a 34 kDa protein denoted as VP7, which was determined to be a viral outer capsid protein (OCP). To obtain expressed OCP in vitro, a full length VP7 gene was produced by RT-PCR amplification, and the amplified fragment was cloned into T7 promoted prokaryotic expression vector pRSET. The recombinant plasmid,which was named as pR/GCRV-VP7,was then transformed into E.coli BL21 host cells. The data indicated that the expressed recombinant was in frame with the N-terminal fusion peptide. The over-expressed fusion protein was produced by inducing with IPTG, and its molecular weight was about 37kDa, which was consistent with its predicted size. In addition, the fusion protein was produced in the form of the inclusion body with their yield remaining steady at more than 60% of total bacterial protein. Moreover,the expressed protein was able to bind immunologically to anti-his-tag monoclonal antibody (mouse) and anti-GCRV serum (rabbit). This work provides a research basis for further structure and function studies of GCRV during entry into cells.     

Exuviae eating: a nitrogen meal?

Many insects eat their cast cuticle (exuviae) after moulting. The functional significance of this behaviour has not been addressed experimentally. I tested the hypothesis that exuviae eating constitutes a meal, so the animal recycles its nitrogen content. Nitrogenous compounds (protein and chitin) are major components of the cuticle in Periplaneta americana, accounting for as much as 87% of the total weight. It was found that insects almost invariably ate their exuviae during their larval life. The frequency of the behaviour decreased in newly emerged adults and varied between the sexes, males eating their exuviae less frequently than females. This may be due to the extra nitrogen endowment which females need for reproduction. Aposymbiotic animals, which lack the supply of essential amino acids from endosymbiotic bacteria, always ate their exuviae regardless of sex. When animals were reared on different diets throughout their larval life protein level in the diet correlated with exuviae eating. Animals reared on a low protein diet showed the highest levels of exuviae eating; animals reared on a high protein diet showed the highest levels of exuviae rejection. Analysis of the frass produced after exuviae meals showed that over 58% of the nitrogen present in the exuviae was recycled. This demonstrated that cockroaches digested nitrogenous compounds contained in the cuticle. The possibility that the exuviae meal has other functions is discussed, although the evidence supports a nutritional role.     

From the structure to the function of villin, an actin-binding protein of the brush border

Villin, a calcium-regulated actin-binding protein, modulates the structure and assembly of actin filaments in vitro. It is organized into three domains, the first two of which are homologous. Villin is mainly produced in epithelial cells that develop a brush border and which are responsible for nutrient uptake. Expression of the villin structural gene is precisely regulated during mouse embryogenesis and is restricted in adults, to certain epithelia of the gastrointestinal and urogenital tracts. The function of villin has been assessed by transfecting CV1 cells with a human cDNA encoding wild-type villin or mutant villin. Synthesis of large amounts of villin in cells which do not normally produce this protein induces the growth of microvilli on the cell surface and the redistribution of F-actin, concomitant with the disappearance of stress fibers. The complete villin sequence is required for the morphogenic effect. These results suggest that villin plays a key role in the morphogenesis of microvilli.     

Borrelia burgdorferi small lipoprotein Lp6.6 is a member of multiple protein complexes in the outer membrane and facilitates pathogen transmission from ticks to mice

Borrelia burgdorferi lipoprotein Lp6.6 is a differentially produced spirochete antigen. An assessment of lp6.6 expression covering representative stages of the infectious cycle of spirochetes demonstrates that the gene is solely expressed during pathogen persistence in ticks. Deletion of lp6.6 in infectious B. burgdorferi did not influence in vitro growth, or its ability to persist and induce inflammation in mice, migrate to larval or nymphal ticks or survive through the larval-nymphal molt. However, Lp6.6-deficient spirochetes displayed significant impairment in their ability to transmit from infected ticks to naïve mice, which was restored upon genetic complementation of the mutant with a wild-type copy of lp6.6 , establishing that Lp6.6 plays a role in pathogen transmission from ticks to mammals. Lp6.6 is a subsurface, yet highly abundant, outer membrane antigen. Two-dimensional blue native/SDS-PAGE coupled with liquid chromatography-mass spectrometry (LC-MS/MS) analysis and protein cross-linking studies independently shows that Lp6.6 exists in multiple protein complexes in the outer membrane. We speculate that the function of Lp6.6 is connected to the physiological processes of these membrane complexes. Further characterization of differentially produced membrane antigens and associated protein complexes will likely aid in our understanding of the molecular details of B. burgdorferi persistence and transmission through a complex enzootic cycle.     

Extra terminal residues have a profound effect on the folding and solubility of a Plasmodium falciparum sexual stage-specific protein over-expressed in Escherichia coli.

The presence of extra N- and C- terminal residues can play a major role in the stability, solubility and yield of recombinant proteins. Pfg27 is a 27K soluble protein that is essential for sexual development in Plasmodium falciparum. It was over-expressed using the pMAL-p2 vector as a fusion protein with the maltose binding protein. Six different constructs were made and each of the fusion proteins were expressed and purified. Our results show that the fusion proteins were labile and only partially soluble in five of the constructs resulting in very poor yields. Intriguingly, in the sixth construct, the yield of soluble fusion protein with an extended carboxyl terminus of 17 residues was several fold higher. Various constructs with either N-terminal or smaller C-terminal extensions failed to produce any soluble fusion protein. Furthermore, all five constructs produced Pfg27 that precipitated after protease cleavage from its fusion partner. The sixth construct, which produced soluble protein in high yields, also gave highly stable and soluble Pfg27 after cleavage of the fusion. These results indicate that extra amino acid residues at the termini of over-expressed proteins can have a significant effect on the folding of proteins expressed in E. coli. Our data suggest the potential for development of a novel methodology, which will entail construction of fusion proteins with maltose binding protein as a chaperone on the N-terminus and a C-terminal 'solubilization tag'. This system may allow large-scale production of those proteins that have a tendency to misfold during expression.     

Infulence of hormones and medium composition on the degradation of phosphoenolpyruvate carboxykinase (GTP) and total protein in Reuber H35 cells.

Reuber H35 cells were pulse-labeled with radioactive leucine and the influence of hormones, serum, and amino acids on protein degradation was investigated during a subsequent chase period. Radioactive, immunoprecipitable phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) had a half-life of 5 to 6 hours which was not influenced by either N6, O2-dibutyryl adenosine 3':5'-monophosphate, dexamethasone, or insulin. The rate of phosphoenolpyruvate carboxykinase degradation was the same under steady state conditions as during the approach to a new steady state following hormonal induction or deinduction of the enzyme. Therefore, hormonal regulation of enzyme activity in vivo is the result of changes in the rate of enzyme synthesis. The rate of proteolysis for total cell proteins was increased under nutritional step-down conditions produced by the removal of serum or amino acids, or both, from the medium. This effect was completely prevented by insulin. Cycloheximide and puromycin, but not actinomycin D or cordycepin, inhibited protein degradation under step-down conditions but did not further decrease the basal rate of proteolysis measured in the presence of either insulin or serum plus amino acids. There was a good correlation between changes in proteolysis produced by serum and amino acids and changes in the degradation rate of phosphoenolpyruvate carboxykinase. Also, inhibition of proteolysis with cycloheximide and puromycin was accompanied by a decrease in the degradation rate for enzyme antigen. It is suggested that nutritional step-down leads either to the synthesis or activation of a proteolytic system.