Tumor promoter benzoyl peroxide induces sulfhydryl oxidation in protein kinase C: its reversibility is related to the cellular resistance to peroxide-induced cytotoxicity

Since tumor promoter benzoyl peroxide (BPO) mimics phorbol esters in some aspects, its effects on protein kinase C (PKC) were previously studied. However, in those studies due to the presence of thiol agents in the PKC preparations, the sensitive reaction of BPO with redox-active cysteine residues in PKC was not observed. In this study, by excluding thiol agents present in the purified PKC preparation, low concentrations of BPO modified PKC, resulting in the loss of both kinase activity and phorbol ester binding (IC50 = 0. 2 to 0.5 microM). This modification, which was not dependent on transition metals, was totally blocked by a variety of thiol agents including GSH, which directly reacted with BPO. Substoichiometric amounts of BPO (0.4 mol/mol of PKC) oxidized two sulfhydryls in PKC and inactivated the enzyme which was readily reversed by dithiothreitol. The regulatory domain having zinc thiolate structures supporting the membrane-inserting region provided the specificity for PKC reaction with BPO, which partitioned into the membrane. Unlike H2O2, BPO did not induce the generation of the Ca2+/lipid-independent activated form of PKC. Other redox-sensitive enzymes such as protein kinase A, phosphorylase kinase, and protein phosphatase 2A required nearly 25- to 100-fold higher concentrations of BPO for inactivation. BPO also inactivated PKC in a variety of cell types. In the JB6 (30 P-) nonpromotable cell line and other normal cell lines, where BPO was more cytotoxic, it readily inactivated PKC due to a slow reversibility of this inactivation by the cell. However, in the JB6 (41 P+) promotable cell line, C3H10T1/2 and B16 melanoma cells, where BPO was less cytotoxic, it did not readily inactivate PKC due to a rapid reversibility of this inactivation by an endogenous mechanism. Nevertheless, BPO inactivated PKC at an equal rate in the homogenates prepared from all these cell types. Inclusion of NADPH reversed this inactivation in the homogenates to a different extent, presumably due to a difference in distribution of a protein disulfide reductase, which reverses this oxidative modification. BPO-induced modification of PKC occurred independent of the cellular status of GSH. However, externally added GSH and cell-impermeable thiol agents prevented the BPO-induced modification of PKC. Since BPO readily partitions into membranes, its reaction with redox-cycling thiols of membrane proteins such as PKC may trigger epigenetic events to prevent cytotoxicity, but favor tumor promotion.     

Tyrosine alpha 244 is derivatized when the bovine heart mitochondrial F1-ATPase is inactivated with 5'-p-fluorosulfonylbenzoylethenoadenosine

The bovine heart mitochondrial F1-ATPase (MF1) is inactivated by 5'-p-fluorosulfonylbenzoylethenoadenosine (FSB epsilon A) with pseudo-first order kinetics. The dependence of the rate of inactivation on the concentration of FSB epsilon A revealed an apparent Kd of 0.25 mM. ATP and ADP, and to a lesser extent, ITP and IDP provide partial protection against inactivation by the reagent. Isolation and sequence analysis of major radioactive fragments in peptic or cyanogen bromide digests of MF1 inactivated with [3H]FSB epsilon A indicate that modification of Tyr-alpha 244 is associated with the loss of activity observed. Assessment of the amount of Tyr-alpha 244 derivatized with [3H]FSB epsilon A at specific points during inactivation of the ATPase indicates that maximal inactivation is achieved on modification of this residue in slightly greater than one copy of the alpha subunit. The following characteristics of inactivation of MF1 by FSB epsilon A have also been determined. (a) The rate of inactivation of ITPase activity by FSB epsilon A is 1.4 times greater than that observed for inactivation of ATPase activity under identical conditions. (b) After maximally inactivating the capacity of MF1 to hydrolyze saturating ATP with FSB epsilon A, the modified enzyme retained its capacity to hydrolyze substoichiometric ATP. (c) Inactivation of the ATPase by FSB epsilon A is accelerated by Pi. In each of the above characteristics, MF1 modified by FSB epsilon A resembles enzyme inactivated with 5'-p-fluorosulfonylbenzoyladenosine (FSBA) more than it does enzyme inactivated with 5'-p-fluorosulfonylbenzoylinosine (FSBI). Furthermore, prior inactivation of MF1 with FSBA completely prevents labeling of Tyr-alpha 244 with [3H]FSB epsilon A, whereas prior inactivation of the enzyme with FSBI does not. Since a single catalytic site is modified when FSBI inactivates MF1 whereas three noncatalytic sites are modified when it is maximally inactivated with FSBA, it is concluded that FSB epsilon A also modifies noncatalytic sites.     

Oxidant-induced S-glutathiolation inactivates protein kinase C-alpha (PKC-alpha): a potential mechanism of PKC isozyme regulation

Protein kinase C (PKC) isozymes are subject to inactivation by reactive oxygen species (ROS) through as yet undefined oxidative modifications of the isozyme structure. We previously reported that Cys-containing, Arg-rich peptide-substrate analogues spontaneously form disulfide-linked complexes with PKC isozymes, resulting in isozyme inactivation. This suggested that PKC might be inactivated by oxidant-induced S-glutathiolation, i.e., disulfide linkage of the endogenous molecule glutathione (GSH) to PKC. Protein S-glutathiolation is a reversible oxidative modification that has profound effects on the activity of certain enzymes and binding proteins. To directly examine whether PKC could be inactivated by S-glutathiolation, we used the thiol-specific oxidant diamide because its oxidant activity is restricted to induction of disulfide bridge formation. Diamide weakly inactivated purified recombinant cPKC-alpha, and this was markedly potentiated to nearly full inactivation by 100 microM GSH, which by itself was without effect on cPKC-alpha activity. Diamide inactivation of cPKC-alpha and its potentiation by GSH were both fully reversed by DTT. Likewise, GSH markedly potentiated diamide inactivation of a PKC isozyme mixture purified from rat brain (alpha, beta, gamma, epsilon, zeta) in a DTT-reversible manner. GSH potentiation of diamide-induced cPKC-alpha inactivation was associated with S-glutathiolation of the isozyme. cPKC-alpha S-glutathiolation was demonstrated by the DTT-reversible incorporation of [(35)S]GSH into the isozyme structure and by an associated change in the migration position of cPKC-alpha in nonreducing SDS-PAGE. Diamide treatment of NIH3T3 cells likewise induced potent, DTT-reversible inactivation of cPKC-alpha in association with [(35)S] S-thiolation of the isozyme. Taken together, the results indicate that PKC isozymes can be oxidatively inactivated by S-thiolation reactions involving endogenous thiols such as GSH.     

Effects of the Antiandrogen Flutamide on the Expression of Protein Kinase C Isoenzymes in LNCaP and PC3 Human Prostate Cancer Cells

Flutamide is a nonsteroidal antiandrogen that is frequently used for total androgen blockage in the treatment of advanced prostate cancer. We investigated the effect of this antiandrogen on the expression of protein kinase C (PKC) isoenzymes (alpha, beta1, epsilon, zeta) that are involved in cell growth, apoptosis and neoplastic transformation. Androgen-dependent (LNCaP) and independent (PC3) human prostate cancer cells were cultured in a medium that contained fetal bovine serum (FBS) or charcoal-stripped serum (CSS) and treated with 10 microM flutamide. The expression of PKC isoenzymes and the androgen receptor (AR) were analyzed by Western blot and RT-PCR, respectively. Serum steroids differentially regulate the expression of PKC isoenzymes in LNCaP and PC3 cells. Flutamide up-regulated the expression of alpha, beta1 and zeta, but not epsilon, PKC isoenzymes in CSS-LNCaP cells. These results were not homogeneously reproduced in the presence of androgens. We observed an opposite effect of flutamide, compared to CSS, on PKCbeta1 isoform expression in CSS-LNCaP suggesting that this antiandrogen exerts an agonistic effect. In PC3 cells flutamide potentiated the expression of the four PKC isoenzymes in almost all conditions tested (FBS- and CSS-cultured cells). Such effect of flutamide in PC3 cells is independent of AR since no expression of AR was detected. These results provide new evidence on antagonistic/agonistic responses of prostate cancer cells to antiandrogen drugs that are widely used in therapy and show that flutamide can elicit responses in prostate cancer cells that do not express AR.     

Regulation of the expression of protein kinase C isoenzymes in rat ventral prostate: effects of age,castration and flutamide treatment

Protein kinase C (PKC) isoenzymes are involved in cell function, growth, apoptosis and neoplastic transformation in the prostate gland. We detected by means of Western blot the expression of the classical alpha and beta1, the novel epsilon and the atypical zeta isoforms of PKC in ventral prostates from rats with different extents of plasma testosterone levels and/or androgen imprinting on the gland. The expression of the four isoforms decreased in 5-day castrated rats showing apoptotical regression of the gland and a drastic reduction of circulating testosterone. However, the expression of PKC isoenzymes (alpha, beta1, epsilon ) increased in prostates from pubertal (35-days old) rats that are characterized by relatively low but extremely bioactive testosterone plasma levels. Treatment of adult rats for 14 days with flutamide (daily s.c. injection of 15 mg/Kg B.W.) resulted in increased expression of the four isoenzymes; it occurred in the presence of increased (normal rats) or drastically reduced (rats castrated after 9 days of flutamide administration) levels of plasma testosterone conceivably through a direct effect of this nonsteroidal antiandrogen on prostate cells. Measurements of PKC(alpha) activity were in agreement with the observations on protein expression and showed that flutamide (that is extensively used in the treatment of advanced prostate cancer) elicits some impairment in the mechanisms of translocation of this isoform from the cytosol to the membrane. Thus, in addition to the possibility of direct effects of flutamide upon the rat prostate, we present evidence that the levels of circulating androgens and/or their bioactivity in the gland regulate the expression of various important PKC isoforms.     

Biphasic effect of protein kinase C activators on spontaneous and glucocorticoid-induced apoptosis in primary mouse thymocytes

Spontaneous and glucocorticoid (fluocinolone acetonide, FA)-induced apoptosis of primary mouse thymocytes was inhibited by protein kinase C (PKC) activators such as bryostatin-1 and phorbol ester 12-O-tetradecanoyl-phorbol-13 acetate (TPA) within the first 2-4 h of incubation but was enhanced upon prolonged treatment. Only the anti-apoptotic but not the pro-apoptotic effect of TPA was completely suppressed by the PKC inhibitor Goe 6983 and moderately inhibited by Goe 6976. Immunoblot analysis revealed distinct PKC alpha, beta, delta, eta, theta, mu and zeta signals, a very faint PKCepsilon and no PKCgamma signal. Upon prolonged TPA treatment all PKC isoenzymes became downregulated, albeit at different rates (PKCdelta>alpha>mu>beta,theta>eta,zeta). No significant generation of caspase-derived catalytic PKC fragments, as found to be produced upon induction of apoptosis and to be pro-apoptotic in other systems, was observed in FA- or TPA-treated thymocytes. It is concluded that the early anti-apoptotic effect of TPA depends on the activation of n-type PKC isoenzymes, whereas stimulation of spontaneous and FA-induced apoptosis by TPA ensues, at least partially, from a downregulation (or inactivation) of anti-apoptotic PKC species, i.e. in primary thymocytes PKC activation is primarily involved in a negative regulation of apoptosis.     

Conjugated linoleic acid inhibits proliferation and induces apoptosis of normal rat mammary epithelial cells in primary culture.

The trace fatty acid conjugated linoleic acid (CLA) inhibits rat mammary carcinogenesis when fed prior to carcinogen during pubertal mammary gland development or during the promotion phase of carcinogenesis. The following studies were done to investigate possible mechanisms of these effects. Using a physiological model for growth and differentiation of normal rat mammary epithelial cell organoids (MEO) in primary culture, we found that CLA, but not linoleic acid (LA), inhibited growth of MEO and that this growth inhibition was mediated both by a reduction in DNA synthesis and a stimulation of apoptosis. The effects of CLA did not appear to be mediated by changes in epithelial protein kinase C (PKC) since neither total activity nor expression nor localization of PKC isoenzymes alpha, beta II, delta, epsilon, eta, or zeta were altered in the epithelium of CLA-fed rats. In contrast, PKCs delta, epsilon, and eta were specifically upregulated and associated with a lipid-like, but acetone-insoluble, fibrillar material found exclusively in adipocytes from CLA-fed rats. Taken together, these observations demonstrate that CLA can act directly to inhibit growth and induce apoptosis of normal MEO and may thus prevent breast cancer by its ability to reduce mammary epithelial density and to inhibit the outgrowth of initiated MEO. Moreover, the changes in mammary adipocyte PKC expression and lipid composition suggest that the adipose stroma may play an important in vivo role in mediating the ability of CLA to inhibit mammary carcinogenesis.     

Expression and activity of protein kinase C isoenzymes during normal and abnormal murine palate development

Protein kinase C (PKC) plays a critical role in signal transduction, mediating various cellular events critical for normal development, including that of the palate. In vivo and in vitro studies suggest the relevance of the inhibition of PKC by the mycotoxin, secalonic acid D (SAD), to its induction of cleft palate (CP) in mice. In the present study, temporal and spatial expression and the activity of various PKC isoenzymes were studied in the control and SAD-exposed murine embryonic palate during gestational days (GD) 12-14.5 by western blotting, immunohistochemistry, and phosphotransfer assay. The Ca2+-dependent isoenzymes, PKC alpha and PKC betaII, showed significant expression on GD 12.0, which gradually decreased through GD 14.5, whereas PKC betaI and PKC gamma were negligible throughout. All Ca2+-independent isoenzymes (epsilon, delta, and zeta) were expressed more abundantly and, in contrast to the Ca2+-dependent ones, progressively increased with age. SAD failed to alter this pattern of expression but enhanced the phosphorylation of PKC epsilon throughout development. Immunohistochemical analysis revealed an isoenzyme-specific distribution of PKC between the epithelium and mesenchyme. As expected, SAD significantly inhibited the total Ca2+-dependent PKC activity in palatal extracts. Although total Ca2+-independent PKC activity in palatal extracts was unaffected by SAD, individual pure isoenzymes were either selectively inhibited (PKC zeta), stimulated (PKC delta), or unaffected (PKC epsilon) by SAD. These results show that PKC isoenzymes exhibit dynamic temporal and spatial patterns of expression and activity in the developing palate and that the induction of CP by SAD is associated with an alteration in their activation and/or activity.     

The redox interconversion mechanism of Saccharomyces cerevisiae glutathione reductase

The changes undergone by pure yeast glutathione reductase during redox interconversion have been studied. Both the active and inactive forms of the enzyme had similar molecular masses, suggesting that the inactivation is probably due to intramolecular modification(s). The glutathione reductase and transhydrogenase activities were similarly inactivated by NADPH and reactivated by GSH, while the diaphorase activity remained unaltered during redox interconversion of glutathione reductase. These results suggest that the inactivation site could be located far from the NADPH-binding site, although interfering with transhydrogenase activity, perhaps by conformational changes. The inactivation of glutathione reductase by 0.2 mM NADPH at pH 8 was paralleled by a gradual decrease in the absorbance at 530 nm and a simultaneous increase in the absorbance at 445 nm, while the reactivation promoted by GSH was initially associated with reversal of these spectral changes. The inactive enzyme spectrum retained some absorbance between 500 nm and 700 nm, showing a shoulder at 580-600 nm. Upon treatment of the enzyme with NADPH at pH 6.5 the spectrum remained unchanged, while no redox inactivation was observed under these conditions. It is suggested that the redox inactivation could be associated with the disappearance of the charge-transfer complex between the proximal thiolate and oxidized FAD in the two-electron-reduced enzyme. The inactive enzyme was reactivated by low GSSG concentrations, moderate dithiol concentrations, and high monothiol concentrations. These results and the spectral changes described above support the hypothesis attributing the redox interconversion to formation/disappearance of an erroneous disulfide between one of the half-cystines located at the GSSG-binding site and another cysteine nearby.     

Reversible oxidative activation and inactivation of protein kinase C by the mitogen/tumor promoter periodate.

The oxidant mitogen/tumor promoter, periodate, was used to selectively modify either the regulatory domain or the catalytic domain of protein kinase C (PKC) to induce oxidative activation or inactivation of PKC, respectively. Periodate, at micromolar concentrations, modified the regulatory domain of PKC as determined by the loss of ability to stimulate kinase activity by Ca2+/phospholipid, and also by the loss of phorbol ester binding. This modification resulted in an increase in Ca2+/phospholipid-independent kinase activity (oxidative activation). However, at higher concentrations (greater than 100 microM) periodate also modified the catalytic domain, resulting in complete inactivation of PKC. The oxidative modification induced by low periodate concentrations (less than 0.5 mM) was completely reversed by a brief treatment with 2 mM dithiothreitol. In this aspect, the modification induced by periodate was different from that of the previously reported irreversible modification of PKC induced by H2O2. However, the inactivation of PKC induced by periodate at concentrations greater than 1 mM was not reversed by dithiothreitol. Among the phospholipids and ligands of the regulatory domain tested, only phosphatidylserine protected the regulatory domain from oxidative modification. In the presence of phosphatidylserine, the catalytic site was selectively modified by periodate, resulting in formation of a form of PKC that exhibited phorbol ester binding but not kinase activity. Both reversible and irreversible oxidative activation and inactivation of PKC also were observed in intact cells treated with periodate. Taken together these results suggest that periodate, by virtue of having a tetrahedral structure, binds to the phosphate-binding regions present within the phosphatidylserine-binding site of the regulatory domain and the ATP-binding site of the catalytic domain, and modifies the vicinal thiols present within these sites. This results in the formation of intramolecular disulfide bridge(s) within the regulatory domain or catalytic domain leading to either reversible activation or inactivation of PKC, respectively. Thus, oxidant mitogen/tumor promoters such as periodate may be able to bypass normal transmembrane signalling systems to directly activate pathways involved in cellular regulation.     

Tissue and cellular distribution of the extended family of protein kinase C isoenzymes

Polyclonal isoenzyme-specific antisera were developed against four calcium-independent protein kinase C (PKC) isoenzymes (delta, epsilon, epsilon', and zeta) as well as the calcium-dependent isoforms (alpha, beta I, beta II, and gamma). These antisera showed high specificities, high titers, and high binding affinities (3-370 nM) for the peptide antigens to which they were raised. Each antiserum detected a species of the predicted molecular weight by Western blot that could be blocked with the immunizing peptide. PKC was sequentially purified from rat brain, and the calcium-dependent forms were finally resolved by hydroxyapatite chromatography. Peak I reacted exclusively with antisera to PKC gamma, peak II with PKC beta I and -beta II, and peak III with PKC alpha. These same fractions, however, were devoid of immunoreactivity for the calcium-independent isoenzymes. The PKC isoenzymes demonstrated a distinctive tissue distribution when evaluated by Western blot and immunocytochemistry. PCK delta was present in brain, heart, spleen, lung, liver, ovary, pancreas, and adrenal tissues. PKC epsilon was present in brain, kidney, and pancreas, whereas PKC epsilon' was present predominantly in brain. PKC zeta was present in most tissues, particularly the lung, brain, and liver. Both PKC delta and PKC zeta showed some heterogeneity of size among the different tissues. PKC alpha was present in all organs and tissues examined. PKC beta I and -beta II were present in greatest amount in brain and spleen. Although the brain contained the most PKC gamma immunoreactivity, some immunostaining was also seen in adrenal tissue. These studies provide the first evidence of selective organ and tissue distributions of the calcium-independent PKC isoenzymes.     

Potent direct inhibition of mammalian phospholipase D isoenzymes by calphostin-c

Calphostin-c inhibits protein kinase C (PKC) isoenzymes by covalent modification of the lipid binding regulatory domain. Exposure of cells to calphostin-c elicits PKC independent effects including disruption of intracellular transport, growth inhibition, and stimulation of apoptosis suggesting actions at additional targets. Phospholipase D (PLD) enzymes are targets for activation by PKC. We have investigated the PKC isoenzyme selectivity for activation of two mammalian PLD enzymes, PLD1 and PLD2, by PKC. We examined the sensitivity of this process to widely used PKC inhibitors and report the surprising finding that calphostin-c is a potent direct inhibitor of PLD1 and PLD2. In vitro, calphostin-c inhibits activity of both PLD1 and PLD2 with an IC(50) of approximately 100 nM. Inhibition is not overcome by protein and lipid activators of these enzymes and does not involve blockade of phosphatidylinositol 4,5-bisphosphate-dependent PLD binding to substrate containing liposomes. Studies using a series of deletion and point mutants of the enzymes suggest that calphostin-c targets the PLD catalytic domain. Inhibition of PLD by calphostin-c in vitro involves stable and apparently irreversible modification of the enzyme. Activity of both PLD1 and PLD2 can be inhibited by calphostin-c treatment of intact cells in a manner that is independent of upstream actions of PKC. Our results suggest that inhibition of PLD1 and PLD2 may explain some of the PKC-independent effects of calphostin-c observed when the compound is applied to intact cells.     

Characterization of C2-ceramide-resistant HL-60 subline (HL-CR): involvement of PKC delta in C2-ceramide resistance

We have established a C2-ceramide-resistant HL-60 subline (HL-CR). HL-CR cells were resistant not only to C2-ceramide but also to various anticancer drugs. HL-CR cells did not respond to differentiation-inducing reagents including 1alpha,25-dihydroxyvitamin D(3), retinoic acid, and 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA induced apoptosis in HL-CR cells much slower than in parental HL-60 cells. As it was reported that PKC isozymes were involved in C2-ceramide-induced apoptosis, we investigated the role of PKC isozymes in C2-ceramide resistance in HL-CR cells. The protein level of PKC delta was lower in HL-CR cells than in parental HL-60 cells, whereas the levels of PKC alpha, betaI, epsilon, and zeta were rather higher in HL-CR cells than in parental cells. Translocation of PKC delta from membrane to cytosol was induced by C2-ceramide in HL-CR cells as well as in wild-type HL-60 cells. Furthermore, overexpression of PKC delta in HL-CR cells potentiated C2-ceramide- and TPA-induced apoptosis and growth inhibition. These results suggest a role for ceramide in apoptosis and differentiation in HL-60 cells, and also suggest that PKC delta might be involved in ceramide- and TPA-induced apoptosis.     

PKC epsilon controls the traffic of beta1 integrins in motile cells

Protein kinase C (PKC) has been implicated in beta 1 integrin-mediated cell migration. Expression of the novel PKC isoform, PKC epsilon, in PKC epsilon(-/-) cells is shown here to stimulate directional migration of cells towards beta 1 integrin substrates in a manner dependent on PKC catalytic activity. On PKC inhibition, integrin beta 1 and PKC epsilon become reversibly trapped in a tetraspanin (CD81)-positive intracellular compartment, correlating with reduced haptotaxis. Immunofluorescence and pulse labelling studies indicate that this is a previously uncharacterized recycling compartment trapped by inhibition of PKC. Electron microscopy demonstrated the co-localization of PKC epsilon and integrin beta 1 on the vesicular membranes. Finally, using a reconstituted in vitro system, the dissociation of PKC epsilon from these vesicles is shown to be dependent on both the presence of cytosolic components and energy, and on PKC catalytic activity. The evidence presented indicates that PKC epsilon controls an internal traffic step that under uninhibited conditions permits the recycling of beta 1 integrin, contributing to cell motility.     

Activation of PKC is sufficient to induce an apoptotic program in salivary gland acinar cells

Accumulating evidence suggests that specific isoforms of PKC may function to promote apoptosis. We show here that activation of the conventional and novel isoforms of PKC with 12-O-tetradecanoyl phorbol-13- ester (TPA) induces apoptosis in salivary acinar cells as indicated by DNA fragmentation and activation of caspase-3. TPA-induced DNA fragmentation, caspase-3 activation, and morphologic indicators of apoptosis, can be enhanced by pretreatment of cells with the calpain inhibitor, calpeptin, prior to the addition of TPA. Analysis of PKC isoform expression by immunoblot shows that TPA-induced downregulation of PKC alpha and PKC delta is delayed in cells pre-treated with calpeptin, and that this correlates with an increase of these isoforms in the membrane fraction of cells. TPA-induced apoptosis is accompanied by biphasic activation of the c-jun-N-terminal kinase (JNK) pathway and inactivation of the extracellular regulated kinase (ERK) pathway. Expression of constitutively activated PKC alpha or PKC delta, but not kinase negative mutants of these isoforms, or constitutively activated PKC epsilon, induces apoptosis in salivary acinar cells, suggesting a role for these isoforms in TPA-induced apoptosis. These studies demonstrate that activation of PKC is sufficient for initiation of an apoptotic program in salivary acinar cells. Cell Death and Differentiation (2000) 7, 1200 - 1209.     

Arginine and lysine residues as NADH-binding sites in NADH-nitrate reductase from spinach.

Chemical modifications of spinach leaf nitrate reductase, and its 28,000 M(r) fragment with phenylglyoxal, 2,3-butanedione and pyridoxal phosphate reduce the catalytic activity of the enzyme. The kinetics of the modification indicate a rapid inactivation followed by a slower rate of inactivation. NADH-nitrate reductase, NADH-cytochrome c reductase and NADH-ferricyanide reductase activities of the nitrate reductase complex are inactivated at a faster rate when compared to the loss of FMNH2-nitrate reductase and reduced methyl viologen (MVH)-nitrate reductase activities. NADH protects the inactivation of NADH-ferricyanide reductase activity of the 28,000 M(r) fragment of nitrate reductase. These data suggest that nitrate reductase contains active sites of arginine and lysine residues that are involved in the NADH binding site of the enzyme.     

Effect of ageing on the expression of protein kinase C and its activation by 1,25(OH)2-vitamin D3 in rat skeletal muscle

To characterize age-induced effects on muscle protein kinase C (PKC) and its regulation by the steroid hormone 1,25(OH)2-vitamin D3 [1,25(OH)2D3], changes in PKC activity and the expression and translocation of the specific PKC conventional isoforms alpha and beta, novel isoforms delta, epsilon, and theta and atypical isoform zeta were studied in homogenates and subcellular fractions from skeletal muscle of young (3 months) and aged (24 months) rats treated in vitro with 1,25(OH)2D3. The hormone (10(-9) M) increased total and membrane PKC activity, within 1 min, and these effects were completely blunted in muscle from aged rats. The presence of PKC isoenzymes was shown by Western blot analysis with the use of specific antibodies. The expression of PKC alpha, beta and delta was greatly diminished in old rats, whereas age-related changes were less pronounced in the isoforms epsilon, theta and zeta. After a short exposure (1 min) of muscle to 1,25(OH)2D3, increased amounts of PKC alpha and beta in muscle membranes and reverse translocation (from membrane to cytosol) of PKC epsilon were observed only in young animals. The data indicate that, in rat muscle, ageing impairs calcium-dependent PKC (alpha and beta) and calcium-independent PKC (delta, epsilon, theta and zeta) signal transduction pathways under selective regulation by 1,25(OH)2D3.     

Affinity labeling of phosphoglycerate kinase by 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine

Yeast phosphoglycerate kinase is irreversibly inactivated upon incubation with 5'-[p-(fluorosulfonyl)-benzoyl]-1-N6-ethenoadenosine (5'-FSB epsilon A), an analogue to the nucleotide substrate. Marked protection against inactivation occurs with MgATP, ATP, MgADP, ADP, and 3-phosphoglycerate, suggesting that a part of the catalytic center is modified. The time dependence of the inactivation is characterized by a nonlinear kinetic profile. Curve fitting of various models for ligand binding to the enzyme suggested a two-site model. Modification of one of the sites appears to protect the catalytically essential site from modification. Stoichiometric studies show that the relationship between moles of 5'-FSB epsilon A incorporated per mole of enzyme and the residual enzymatic activity also shows nonlinear behavior. An extrapolated value of 1.5 mol of bound label/mol of enzyme corresponds to complete inactivation. The apparent overall pseudo first-order rate constant for the reaction between phosphoglycerate kinase and 5'-FSB epsilon A, as well as the separate rate constants for the modification, exhibit saturation behavior with respect to the concentration of 5'-FSB epsilon A, indicative of a rapid reversible binding of the reagent to the enzyme prior to modification.     

PKC alpha regulates the hypertrophic growth of cardiomyocytes through extracellular signal-regulated kinase1/2 (ERK1/2)

Members of the protein kinase C (PKC) isozyme family are important signal transducers in virtually every mammalian cell type. Within the heart, PKC isozymes are thought to participate in a signaling network that programs developmental and pathological cardiomyocyte hypertrophic growth. To investigate the function of PKC signaling in regulating cardiomyocyte growth, adenoviral-mediated gene transfer of wild-type and dominant negative mutants of PKC alpha, beta II, delta, and epsilon (only wild-type zeta) was performed in cultured neonatal rat cardiomyocytes. Overexpression of wild-type PKC alpha, beta II, delta, and epsilon revealed distinct subcellular localizations upon activation suggesting unique functions of each isozyme in cardiomyocytes. Indeed, overexpression of wild-type PKC alpha, but not betaI I, delta, epsilon, or zeta induced hypertrophic growth of cardiomyocytes characterized by increased cell surface area, increased [(3)H]-leucine incorporation, and increased expression of the hypertrophic marker gene atrial natriuretic factor. In contrast, expression of dominant negative PKC alpha, beta II, delta, and epsilon revealed a necessary role for PKC alpha as a mediator of agonist-induced cardiomyocyte hypertrophy, whereas dominant negative PKC epsilon reduced cellular viability. A mechanism whereby PKC alpha might regulate hypertrophy was suggested by the observations that wild-type PKC alpha induced extracellular signal-regulated kinase1/2 (ERK1/2), that dominant negative PKC alpha inhibited PMA-induced ERK1/2 activation, and that dominant negative MEK1 (up-stream of ERK1/2) inhibited wild-type PKC alpha-induced hypertrophic growth. These results implicate PKC alpha as a necessary mediator of cardiomyocyte hypertrophic growth, in part, through a ERK1/2-dependent signaling pathway.     

Reactions of the Neurospora crassa nitrate reductase with NAD(P) analogs.

The assimilatory NADPH-nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.3) from Neurospora crassa is competitively inhibited by 3-aminopyridine adenine dinucleotide (AAD) and 3-aminopyridine adenine dinucleotide phosphate (AADP) which are structural analogs of NAD and NADP, respectively. The amino group of the pyridine ring of AAD(P) can react with nitrous acid to yield the diazonium derivative which may covalently bind at the NAD(P) site. As a result of covalent attachment, diazotized AAD(P) causes time-dependent irreversible inactivation of nitrate reductase. However, only the NADPH-dependent activities of the nitrate reductase, i.e. the overall NADPH-nitrate reductase and the NADPH-cytochrome c reductase activities, are inactivated. The reduced methyl viologen- and reduced FAD-nitrate reductase activities which do not utilize NADPH are not inhibited. This inactivation by diazotized AADP is prevented by 1 mM NADP. The inclusion of 1 muM FAD can also prevent inactivation, but the FAD effect differs from the NADP protection in that even after removal of the exogenous FAD by extensive dialysis or Sephadex G-25 filtration chromatography, the enzyme is still protected against inactivation. The FAD-generated protected form of nitrate reductase could again be inactivated if the enzyme was treated with NADPH, dialyzed to remove the NADPH, and then exposed to diazotized AADP. When NADP was substituted for NADPH in this experiment, the enzyme remained in the FAD-protected state. Difference spectra of the inactivated nitrate reductase demonstrated the presence of bound AADP, and titration of the sulfhydryl groups of the inactivated enzyme revealed that a loss of accessible sulfhydryls had occurred. The hypothesis generated by these experiments is that diazotized AADP binds at the NADPH site on nitrate reductase and reacts with a functional sulfhydryl at the site. FAD protects the enzyme against inactivation by modifying the sulfhydryl. Since NADPH reverses this protection, it appears the modifications occurring are oxidation-reduction reactions. On the basis of these results, the physiological electron flow in the nitrate reductase is postulated to be from NADPH via sulfhydryls to FAD and then the remainder of the electron carriers as follows: NADPH leads to -SH leads to FAD leads to cytochrome b-557 leads to Mo leads to NO-3.