Demonstration of a direct effect of inhibitors of the degradation of receptor-bound human choriogonadotropin on the steroidogenic pathway.

Results presented in the previous paper (Ascoli, M., and Puett, D. (1978) J. Biol. Chem. 253, 7832--7838) show that the degradation of receptor-bound 125I-labeled human choriogonadotropin can be inhibited by chloroquine, protease inhibitors, and metabolic inhibitors. These compounds were also shown to inhibit gonadotropin-stimulated steroidogenesis. It is reported herein that these inhibitors also block the stimulation of steroidogenesis by both cholera toxin and 8-Br-adenosine 3':5'-monophosphate, thus showing that they are not specific for the hormonal stimuli. These results, taken together with previous observations that show that NH2Cl can block hormone degradation without inhibiting hormone-stimulated steroidogenesis, strongly suggest that the degradation of choriogonadotropin is not required for its stimulatory action on progesterone production.     

Inhibition of gonadotropin-responsive adenylate cyclase in MA-10 Leydig tumor cells by epidermal growth factor

We have previously shown that mouse epidermal growth factor (mEGF) attenuates the increase in intracellular cAMP provoked by human choriogonadotropin (hCG) in MA-10 Leydig tumor cells (Ascoli, M., Euffa, J., and Segaloff, D. L. (1987) J. Biol. Chem. 262, 9196-9203). The studies presented herein were designed to investigate the mechanism(s) responsible for this phenomenon. We show that mEGF attenuates the increase in cAMP accumulation provoked by hCG primarily, if not entirely, by inhibiting adenylate cyclase activity. This phenomenon has some specificity for the agonist used, but it is not cell-specific. Thus, mEGF inhibited hCG-activated adenylate cyclase in MA-10 cells and in rat luteal cells but had no effect on the forskolin-activated enzyme in MA-10 cells or the isoproterenol-activated enzyme in rat luteal cells.     

Creatine phosphate inhibition of heart lactate dehydrogenase and muscle pyruvate kinase is due to a contaminant

Previously reported inhibitions of heart lactate dehydrogenase (Guppy, M., and Hochachka, P.W. (1978) J. Biol. Chem. 253, 8465-8469) and muscle pyruvate kinase (Kemp, R.G. (1973) J. Biol. Chem. 248, 3963-3967) by creatine phosphate are due to oxalate which is a contaminant found in some commercial preparations of creatine phosphate.     

Migration of 40 S ribosomal subunits on messenger RNA when initiation is perturbed by lowering magnesium or adding drugs.

Migration of 40 S ribosomal subunits on messenger RNA, detected previously in experiments using the antibiotic edeine (Kozak, M., and Shatkin, A.J. (1978) J. Biol. Chem. 253, 6568-6577) has now been observed in the presence of other inhibitors of initiation. 40 S subunit migration has been detected in both wheat germ and reticulocyte lysates treated with edeine, pactamycin, or sodium fluoride. The variety of structurally unrelated inhibitors that mediate this effect argues against the interpretation that migration is a drug-induced artifact. Indeed, limited migration of 40 S ribosomes occurs upon simply lowering the magnesium concentration, in the absence of inhibitors. Thus, migration seems to be an inherent property of 40 S ribosomal subunits and might be involved in the mechanism by which eukaryotic ribosomes select initiation sites in messenger RNA.     

Chemical synthesis and some properties of 6-substituted flavins

A number of derivatives of riboflavin and of 3-methyllumiflavin substituted in the 6 position have been synthesized starting with 6-nitro flavins, reduction to the 6-amino flavin, and diazotization, followed by reaction with the appropriate nucleophile. The absorption spectra, oxidation-reduction potentials, and the electron spin resonance spectra of the radical cationic forms of several of these synthetic compounds have been determined, including 6-S-cysteinyl-3-methyllumiflavin and 6-S-cysteinylriboflavin. The latter has been shown to be identical with the dephosphorylated form of the aminoacyl flavin isolated from trimethylamine dehydrogenase [Steenkamp, D. J., Kenney, W. C. & Singer, T. P. (1978) J. Biol. Chem. 253, 2812-2817; Steenkamp, D. J., McIntire, W., & Kenney, W. C. (1978) J. Biol. Chem. 253, 2818-2824] in regard to absorption specturm, photochemical properties, and mobility in high-voltage electrophoresis and in thin-layer chromatography. An unusually pronounced interaction between the amino group and the isoalloxazine ring system was deduced from the absorption spectra of 6-amino-3-methyllumiflavin and 6-aminoriboflavin.     

G alpha minigenes expressing C-terminal peptides serve as specific inhibitors of thrombin-mediated endothelial activation

The C termini of G protein alpha subunits are critical for binding to their cognate receptors, and peptides corresponding to the C terminus can serve as competitive inhibitors of G protein-coupled receptor-G protein interactions. This interface is quite specific as a single amino acid difference annuls the ability of a G alpha(i) peptide to bind the A(1) adenosine receptor (Gilchrist, A., Mazzoni, M., Dineen, B., Dice, A., Linden, J., Dunwiddie, T., and Hamm, H. E. (1998 ) J. Biol. Chem. 273, 14912--14919). Recently, we demonstrated that a plasmid minigene vector encoding the C-terminal sequence of G alpha(i) could specifically inhibit downstream responses to agonist stimulation of the muscarinic M(2) receptor (Gilchrist, A., Bunemann, M., Li, A., Hosey, M. M., and H. E. Hamm (1999) J. Biol. Chem. 274, 6610--6616). To selectively antagonize G protein signal transduction events and determine which G protein underlies a given thrombin-induced response, we generated minigene vectors that encode the C-terminal sequence for each family of G alpha subunits. Minigene vectors expressing G alpha C-terminal peptides (G alpha(i), G alpha(q), G alpha(12), and G alpha(13)) or the control minigene vector, which expresses the G alpha(i) peptide in random order (G(iR)), were systematically introduced into a human microvascular endothelial cell line. The C-terminal peptides serve as competitive inhibitors presumably by blocking the site on the G protein-coupled receptor that normally binds the G protein. Our results not only confirm that each G protein can control certain signaling events, they emphasize the specificity of the G protein-coupled receptor-G protein interface. In addition, the C-terminal G alpha minigenes appear to be a powerful tool for dissecting out the G protein that mediates a given physiological function following thrombin activation.     

The ubiquitin-activating enzyme, E1, is required for stress-induced lysosomal degradation of cellular proteins.

ts85, a cell line that harbors a mutant thermolabile ubiquitin-activating enzyme, E1, fails to degrade short lived proteins at the restrictive temperature (Ciechanover, A., Finley, D., and Varshavsky, A. (1984) Cell 37, 57-66). However, the involvement of the ubiquitin system in the degradation of long lived proteins (most cellular proteins fall in this category) has not been addressed. In the present study we show that upon shifting the mutant cells to the restrictive temperature, there is no change in the rate of degradation of long lived proteins. In contrast, shifting the wild-type cells (FM3A) to the high temperature is accompanied by a 2-fold increase in the rate of proteolysis of this group of proteins. This heat-induced accelerated degradation can be inhibited completely by NH4Cl and chloroquine. Similarly, exposure of the cells to starvation, a stimulus that activates the autophagic-lysosomal pathway, has no effect on the degradation of long lived proteins in the mutant cells after inactivation of E1. Under the same conditions, the degradation rate in the wild-type cells increases almost 4-fold. Analogous results were obtained using a different cell line that also harbors a thermolabile E1 (ts20 (Kulka, R. G., Raboy, B., Schuster, R., Parag, H. A., Diamond, G., Ciechanover, A., and Marcus, M. (1988) J. Biol. Chem. 263, 15726-15731)). Cycloheximide and 3-methyladenine, known inhibitors of formation of autophagic vacuoles, inhibit the heat-induced accelerated degradation of long lived proteins in wild-type cells. Taken together, the results suggest that 1) heat stress induces enhanced degradation of intracellular proteins; 2) the process occurs most probably in autophagic vacuoles; and 3) activation of ubiquitin is required for the formation of these vacuoles. As there is no change in the basal rate of degradation of intracellular proteins in the mutant cells at the restrictive temperature, it appears that the ubiquitin system is not involved in their breakdown.     

Purification and properties of the manganese superoxide dismutase from the liver of bullfrog, Rana catesbeiana

A manganese superoxide dismutase (Mn-SOD) from the liver of bullfrog, Rana catesbeiana, was purified to electrophoretic homogeneity. The enzyme has a molecular weight of about 84,000 and is composed of four identical subunits, each containing one manganese atom. The amino acid composition of the enzyme is similar to that of Mn-SODs isolated from human and chicken livers, but differs considerably from that of the Escherichia coli enzyme (D. Barra et al. (1984) J. Biol. Chem. 259, 12595-12601; R. A. Weisiger and I. Fridovich (1973) J. Biol. Chem. 248, 3582-3592; H. M. Steinman (1978) J. Biol. Chem. 253, 8708-8720). The N-terminal amino acid is lysine. The sequence of 23 amino acid residues in the N-terminal region was determined. It shows excellent homologies with those of the human and chicken enzymes (H. M. Steinmam and R. L. Hill (1973) Proc. Natl. Acad. Sci. USA 70, 3725-3729; C. Ditlow et al. (1982) Carlsberg Res. Commun. 47, 81-91). The frog liver enzyme is also located exclusively in the mitochondrial matrix. Immunologically the same enzyme is also found in the tadpole liver, in an amount of about one-half of that in the adult bullfrog.     

Modulation of mannose receptor activity by proteolysis.

The binding, internalization and degradation of 200 pM monoiodinated human atrial natriuretic factor-(99-126) (125I-hANF) by cultured bovine aortic endothelial cells (BAECs) were studied at 37 degrees C. 125I-hANF was rapidly cleared from the extracellular medium (t1/2 approximately 10 min), whereas preincubation of the cells in the presence of 20 mM-NH4Cl or 0.2 mM-chloroquine resulted in a significant inhibition of this process. The BAECs rapidly produce three major degradation products of 125I-hANF, namely [125I]iodotyrosine 126 (125I-Y), Arg125-[125I]iodotyrosine126 (125I-RY) and Phe124-Arg125-[125I]iodotyrosine126(125I-FRY), which were detected in the extracellular medium. NH4Cl and chloroquine acted to inhibit the generation of 125I-Y and 125I-RY, but not that of 125I-FRY. Furthermore, excess unlabelled hANF (300 nM) completely blocked the rapid production of 125I-Y and 125I-RY in the first 5 min, but only partially (49%) inhibited the generation of 125I-FRY. Thus, in contrast with our previous findings with cultured smooth-muscle cells [Johnson, Arik & Foster (1989) J. Biol. Chem. 264, 11637-11642], BAECs bind, internalize and rapidly degrade 125I-hANF, resulting in the release of 125I-Y and 125I-RY into the extracellular medium. Similarly to smooth-muscle cells, the BAECs generate 125I-FRY from 125I-hANF via an extracellular proteolytic event. The rapidity of the receptor-mediated process and its sensitivity to NH4Cl and chloroquine suggest that the 125I-hANF is proteolytically processed in the endosomes of BAECs and that its receptors cycle between the cell surface and intracellular stores.     

Nucleotides sequence of the genes for the simian virus 40 proteins VP2 and VP3.

We have determined the nucleotide sequence of the DNA of simian virus 40. The proceeding report (Dhar, R., Reddy, V.B., and Weissman, S.M. (1978) J. Biol. Chem. 253, 612-620) presents the sequence of a portion of the simian virus 40 DNA that overlaps the region encoding the 5' end of the minor structural protein VP2. We report here the sequence of the remainder of the genes for minor structural proteins VP2 and VP3. The results indicate that the mRNA for the two proteins is read in the same phase and the initiation site for VP3 lies within the structural gene of VP2. The codons of the COOH-terminal amino acids of VP2 and VP3 are read in a second phase as the codons of the NH2-terminal amino acids of VP1.     

Cholecystokinin/pancreozymin induces the parallel discharge of digestive enzymes from the in vitro rabbit pancreas.

Considerable controversy has surrounded the question of whether the exocrine pancreas discharges digestive enzymes in a parallel or nonparallel fashion. A recent report (Rothman, S.S., and Wilking, H. (1978) J. Biol. Chem. 253, 3543-3549) claimed that the in vitro rabbit pancreas demonstrated nonparallel enzyme discharge after stimulation with cholecystokinin/pancreozymin, but that parallel discharge followed stimulation with the COOH-terminal octapeptide of cholecystokinin/pancreozymin. It was suggested that the full hormone acted to inhibit chymotrypsinogen secretion while stimulating trypsinogen secretion. Because of the fundamental importance of this question to our understanding of the exocrine secretion of exportable proteins, we have repeated these experiments using the same preparation and stimulant but have observed only parallel enzyme discharge. We conclude that it is unlikely that cholecystokinin/pancreozymin causes the selective inhibition of chymotrypsinogen secretion.     

9-LR-linoleyl hydroperoxide, a novel product from the oxygenation of linoleic acid by type-2 lipoxygenases from soybeans and peas.

Type-2 lipoxygenases from soybeans and peas, which have a pH optimum of 6--7 were examined for oxygenation activity at pH 9.0. The reaction velocity was found to be strongly dependent on substrate concentration. At higher substrate concentrations an inhibitory effect was observed, which is connected with the occurrence of a kinetic lag phase. On incubation of linoleic acid at pH 9.0 with either of these enzymes predominantly 9-LR-hydroperoxy-10-trans,12-cis-octadecadienoic acid is formed. The similarity of the product specificity with that of prostaglandin synthetase is discussed in view of the formation of prostaglandin-like substances by soybean lipoxygenase-2 (Bild, G.S., Bhat, S.G., Ramadoss, C.S. and Axelrod, B. (1978) J. Biol. Chem, 253, 21--23).     

Identification of pseudoisoenzymic subforms of muscle carbonic anhydrase.

Rabbit muscle carbonic anhydrase III, a recently discovered third isoenzyme (possibly muscle specific) of carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) (Register, A.M., Koester, M.K. and Noltmann, E.A. (1978) J. Biol. Chem. 253, 4143--4152) has been subjected to isoelectric focusing. When monomer samples, shown to be homogeneous by both ion-exchange and molecular sieve chromatography, were analyzed by this technique, three subspecies were produced, which were similar in amino acid composition and specific CO2 hydratase activity. In addition to having either monomer or dimer status, the subspecies differed in the extent of oxidation of their sulhydryl groups and in their isoelectric pH values (9.3, 8.8, and 8.4, respectively). Also, the presence of dithiothreitol will affect their relative concentrations. These subforms are therefore designated as pseudoisoenzymes and are considered to be neither genetically nor functionally separate enzyme species.     

Aminoacridines, potent inhibitors of protein kinase C

Acridine orange, acridine yellow G, and related compounds potently inhibited protein kinase C (Ca2+/phospholipid-dependent enzyme) activity and phorbol dibutyrate binding. Inhibition was investigated in vitro using Triton X-100 mixed micellar assays (Hannun, Y. A., Loomis, C. R., and Bell, R. M. (1985) J. Biol. Chem. 260, 10039-10043 and Hannun, Y. A., and Bell, R. M. (1986) J. Biol. Chem. 261, 9341-9347). Inhibition by the acridine derivatives was subject to surface dilution; therefore, the relevant concentration unit is mol % rather than the bulk molar concentration. Fifty percent inhibition of protein kinase C activity occurred at concentrations of these compounds comparable to concentrations of sn-1,2-diacylglycerol (DAG) and phosphatidylserine (PS) required for enzyme activation (i.e. 1-6 mol %). The mechanism of inhibition appeared to be complex: both the catalytic and regulatory sites of protein kinase C were affected. Acridine orange was a competitive inhibitor with respect to MgATP when the catalytic fragment of protein kinase C was employed. Inhibition at the active site was overcome by the addition of Triton X-100 micelles or phospholipid vesicles. When the activity of intact protein kinase C was measured, inhibition was noncompetitive with respect to MgATP. Further kinetic analysis suggested a competitive type of inhibition with respect to PS and DAG implying an interaction of acridine compounds with the regulatory lipid cofactors or with the regulatory domain of protein kinase C. This was further supported by demonstrating inhibition of phorbol dibutyrate binding to both protein kinase C and the lipid-binding domain generated by trypsin hydrolysis. Acridine orange and acridine yellow G also inhibited thrombin-induced 40-kDa phosphorylation in human platelets and phorbol dibutyrate binding to platelets. These effects were also subject to surface dilution. These results suggest that acridine derivatives have multiple interactions with protein kinase C with the predominant effect being inhibition of activation within the regulatory domain of the enzyme. Some of the biologic effects of acridine derivatives including anti-tumor action may occur as a consequence of protein kinase C inhibition.     

Soluble aggregates of the human PiZ alpha 1-antitrypsin variant are degraded within the endoplasmic reticulum by a mechanism sensitive to inhibitors of protein synthesis.

Greater than 85% of the transport-impaired PiZ variant of human alpha 1-antitrypsin is retained within transfected mouse hepatoma cells and is subjected to intracellular degradation (Le, A., Graham, K., and Sifers, R.N. (1990) J. Biol. Chem. 265, 14001-14007). The retained protein undergoes a discrete size reduction that results from the modification of its endoglycosidase H-sensitive oligosaccharides and is inhibited by 1-deoxymannojirimycin. Metabolic poisons and inhibitors of protein synthesis perturb the intracellular degradation of the retained protein but do not affect its size reduction. The ability of metabolic poisons to influence the degradation of the PiZ variant in cells treated with brefeldin A indicates that export of the macromolecule from the endoplasmic reticulum (ER) is not the energy-dependent component of its degradation. Subcellular fractionation experiments have verified that both the size reduction and degradation of the retained PiZ variant occur within the rough ER. Finally, sedimentation velocity centrifugation analysis of radiolabeled cell extracts has indicated that approximately 80% of the PiZ variant consists as soluble aggregates immediately after its synthesis. An inability to detect more extensive aggregation during the retention period supports our previous conclusion that only a small fraction of the macromolecules actually form large insoluble aggregates (Graham, K.S., Le, A., and Sifers, R.N. (1990) J. Biol. Chem. 265, 20463-20468). Overall, these findings indicate that soluble aggregates of the PiZ variant are degraded within the ER by a mechanism sensitive to inhibitors of protein synthesis.     

Evidence for a reciprocal relationship between lipogenesis and ketogenesis in hepatocytes from fed virgin and lactating rats.

Lipogenesis is increased in hepatocytes from fed lactating rats compared with virgin rats. Inhibition of lipogenesis with 5-(tetradecyloxy)-2-furoic acid resulted in increased ketogenesis from endogenous substrate, but not from oleate. Dihydroxyacetone increased ketogenesis from endogenous substrate, but not from oleate. Dihydroxyacetone increased lipogenesis and esterification of [1--14C]oleate and decreased ketogenesis; these changes were reversed by the inhibitor. The reciprocal relationship between lipogenesis and ketogenesis in hepatocytes from fed rats may be due to alterations in [malonyl-CoA] [McGarry, Mannaerts & Foster (1977) J. Clin. Invest. 60, 265--270; Cook, King & Veech (1978) J. Biol. Chem. 253, 2529--2531], but this mechanism is not considered to be sufficient to explain the increased ketogenesis in starvation completely.     

The ATP-independent pathway in red blood cells that degrades oxidant-damaged hemoglobin.

Studies were carried out to characterize further the cytoplasmic ATP- and ubiquitin-independent proteolytic system in red blood cells that degrades hemoglobin damaged by exposure to oxidants (Fagan, J. M., Waxman, L., and Goldberg, A. L. (1986) J. Biol. Chem. 261, 5705-5713). Several proteases were ruled out as having a major role in the degradation of oxidant-treated hemoglobin (Ox-Hb). Acid hydrolases are not active in this process since the degradation of Ox-Hb has a pH optimum between 6 and 8. The calpains are also not involved since inhibitors of cysteine proteases (leupeptin and trans-epoxysuccinyl-L-leucylamido-(3-methyl)butane) did not diminish the increased proteolysis in intact erythrocytes treated with oxidants or in lysates to which Ox-Hb was added. The degradation of Ox-Hb was unaffected by inhibitors of serine and aspartic proteases. Removal of the high M(r) multicatalytic proteinase by immunoprecipitation also did not significantly affect the degradation of Ox-Hb in erythrocyte lysates. The degradation of Ox-Hb was sensitive to metal chelators and sulfhydryl-modifying reagents but not to specific inhibitors of known metalloproteases. Insulin, which is rapidly degraded in lysates, completely blocked the degradation of Ox-Hb. Insulin- and Ox-Hb-hydrolyzing activity was also inhibited following immunoprecipitation of the 100-kDa metalloinsulinase. The metalloinsulinase, which is inhibited by sulfhydryl-modifying reagents and which requires divalent metals, may therefore participate in the degradation of hemoglobin damaged by oxidants in erythrocytes.     

Characterization of human C4a anaphylatoxin

Human C4a anaphylatoxin was isolated from a Cls digest of the fourth component of complement. Isolation required a two-step procedure involving ion-exchange chromatography on CM-Sephadex C-50 and gel filtration on Sephadex G-50. Characterization of C4a indicated it is a highly cationic polypeptide (pI = 9.0-9.5) containing 77 residues with Mr = 8,759. C4a is devoid of tryptophan, histidine, and carbohydrate. Judged by the shape and magnitude of its circular dichroism spectrum, 54% of the polypeptide backbone of C4a assumes an alpha-helical conformation. Partial NH2-terminal sequence determination of C4a revealed a sequence identical with that published by Bolotin et al. (Bolotin, C., Morris, S., Tack, B., and Prahl, J. (1977) Biochemistry 16, 2008-2015) for the NH2 terminus of the alpha-subunit of human C4. Comparison of the NH2-terminal sequence of C4a with the sequences of complement activation fragments C3a (Hugli, T.E. (1975) J. Biol. Chem. 250, 8293-8301) and C5a (Fernandez, H.N., and Hugli, T.E. (1978) J. Biol. Chem, 253-6955-6962) showed that of the first 24 NH2-terminal residues of C4a, 6 were identical with those of C3a (25% homology) and 8 were identical with those of C5a (33% homology). These data represent the first chemical evidence for the existence of an evolutionary relationship among anaphylatoxins C3a, C4a, and C5a, and imply that a similar relationship exists among their precursor proteins.     

Paradoxical effects of protein synthesis inhibitors on uridine uptake in cultured cells: Possible role of uncharged tRNA in regulating metabolism

Previous studies (J. Biol. Chem, 253: 99–105, 1978) showed that thyrotropin-releasing hormone (TRH) acutely stimulated uridine uptake in pituitary cell (GH₄C₁) cultures. Studies on the role of protein synthesis in this response to TRH led to the finding that an inhibitor of ribosomal translation, cycloheximide, also stimulated uridine uptake acutely. Studies reported here attempt to determine the mechanism of cycloheximide action and whether cycloheximide and hormone stimulation of uridine uptake occurred by similar pathways. The experiments presented indicate that: (1) seven inhibitors of ribosomal translation stimulated uridine uptake; (2) in contrast, inhibition of protein synthesis at tRNA aminoacylation resulted in reduced rates of uridine uptake; (3) inhibition of tRNA aminoacylation blocked cycloheximide but not TRH stimulation of uptake; (4) cycloheximide stimulation of uptake was restricted to amino acid-depleted cultures; (5) amino acid supplementation stimulated uridine uptake with a time-course identical to that of cycloheximide; (6) cycloheximide and amino acid supplementation promoted reacylation of cellular tRNAs in amino acid-depleted cultures; and (7) cycloheximide stimulation of uridine uptake resulted from enhanced nucleoside phosphorylation rather than increased uridine transport. We conclude that cycloheximide and amino acid stimulation of uridine phosphorylation may be mediated through a common pathway involving the extent of amino-acylation of cellular tRNAs. Furthermore, cycloheximide and TRH stimulate uridine phosphorylation by pathways that are distinguishable. It is apparent that not all cellular effects of cycloheximde can be attributed solely to inhibition of the synthesis of proteins.