Rapid in vivo inactivation by acivicin of CTP synthetase,carbamoyl-phosphate synthetase II,and amidophosphoribosyltransferase in hepatoma

A single injection of the anti-glutamine drug, acivicin (NSC 163501), in tumor-bearing rats in 30 min decreased the activities of amidophosphoribosyltransferase, carbamoyl-phosphate synthetase II and CTP synthetase to 56, 50 and 7% of those of the controls. By 1 hr the activities were down to 32, 13 and 3% and they remained low for 12 hr, after which they slowly returned towards normal range in 72 hr. The decline of the activity of CTP synthetase (a loss of 80% in 10 min) was the most rapid, and the activity only returned to 60% of the controls by 3 days after the acivicin injection. In the hepatoma the concentrations of ATP and UTP changed little, but those of GTP and CTP rapidly decreased, reaching at the lowest point 32 and 2%, respectively, of control values 2 hr after acivicin; concentrations started to rise at 12 hr, reaching normal levels by 48 hr. The drop in enzyme activities preceded the decline in the pools of GTP and CTP. The behavior of enzyme activities and nucleotide concentrations in the host liver had a pattern similar to that in the hepatoma; however, the changes were less extensive than those in the tumor. The differential response between tumor and liver is attributed, in part at least, to the tissue L-glutamine concentration which in the hepatoma (0.5 mM) was 9 times lower than in the liver (4.5 mM). The selectivity of acivicin action in inhibiting glutamine-utilizing enzymes is also demonstrated by the lack of effect on aspartate carbamoyltransferase, a an enzymic activity which resides in the same complex as that of carbamoyl-phosphate synthetase II. The rapid decline in the activities of glutamine-utilizing enzymes is attributed to an in-activation of the enzymes by acivicin which functions as an active site-directed affinity analog of L-glutamine. The rapid modulation of the enzymic phenotype and ribonucleotide concentrations by acivicin provides a useful tool for elucidating the role of enzymic and nucleotide imbalance in the commitment of cancer cells to replication and in the targeting of anticancer chemotherapy.     

Biochemical basis for the specificity of alloxan inactivation of calmodulin-dependent protein kinase II.

The specificity and biochemical basis of inactivation of calmodulin-dependent protein kinase II by alloxan was studied in dispersed rat brain cells and a partially purified kinase preparation from an insulin-secreting tumor-cell line, RINm5f. When mechanically dispersed rat brain cells were incubated with [32P]-phosphate to label endogenous ATP, depolarization with 44 mM KCl produced a significant (P = 0.03) increase in phosphorylation of endogenous synapsin (132 +/- 8% of basal). Pre-treatment of the brain cells with 1.5 mM alloxan reduced depolarization-sensitive synapsin phosphorylation (109 +/- 5%). Phosphopeptide mapping of depolarization-phosphorylated synapsin showed that alloxan pre-treatment reduced phosphorylation specifically at synapsin sites phosphorylated by calmodulin-dependent protein kinase II. The results demonstrate selective inactivation of calmodulin-dependent protein kinase II activity by alloxan in an intact cell system, which may be useful in the study of the Type II kinase in cells and tissues. Using a partially purified kinase preparation from RINm5f cells, alloxan (100 microM) inactivated 76 +/- 1% calmodulin-dependent protein kinase II activity in 5 min at 37 degrees C. Subsequent incubation with dithiothreitol restored most of the activity. 5,5'-Dithiobis (2-nitrobenzoic acid) (I50 = 2.5 microM) also inactivated the kinase. These results suggested that a sulfhydryl group was involved at the inactivation site. Iodoacetamide (1.0 mM) had no inhibitory effect; however, preincubation with iodoacetamide protected the kinase activity from subsequent inactivation by alloxan. Covalent binding of [14C]-alloxan to calmodulin-dependent protein kinase was demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism for acivicin inactivation of triad glutamine amidotransferases

Acivicin [(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid] was investigated as an inhibitor of the triad glutamine amidotransferases, IGP synthase and GMP synthetase. Nucleophilic substitution of the chlorine atom in acivicin results in the formation of an imine-thioether adduct at the active site cysteine. Cys 77 was identified as the site of modification in the heterodimeric IGPS from Escherichia coli (HisHF) by tryptic digest and FABMS. Distinctions in the glutaminase domains of IGPS from E. coli, the bifunctional protein from Saccharomyces cerevisiae (HIS7), and E. coli GMPS were revealed by the differential rates of inactivation. While the ammonia-dependent turnover was unaffected by acivicin, the glutamine-dependent reaction was inhibited with unit stoichiometry. In analogy to the conditional glutaminase activity seen in IGPS and GMPS, the rates of inactivation were accelerated > or =25-fold when a nucleotide substrate (or analogue) was present. The specificity (k(inact)/K(i)app) for acivicin is on the same order of magnitude as the natural substrate glutamine in all three enzymes. The (alphaS,5R) diastereomer of acivicin was tested under identical conditions as acivicin and showed little inhibitory effect on the enzymes indicating that acivicin binds in the glutamine reactive site in a specific conformation. The data indicate that acivicin undergoes a glutamine amidotransferase mechanism-based covalent bond formation in the presence of nucleotide substrates or products. Acivicin and its (alphaS,5R) diastereomer were modeled in the glutaminase active site of GMPS and CPS to confirm that the binding orientation of the dihydroisoxazole ring is identical in all three triad glutamine amidotransferases. Stabilization of the imine-thioether intermediate by the oxyanion hole in triad glutamine amidotransferases appears to confer the high degree of specificity for acivicin inhibition and relates to a common mechanism for inactivation.     

Uptake and metabolism of glutamine in cultured kidney cells

The metabolism of glutamine was investigated in cultured rat kidney cells. Glutamine utilization and product formation were followed as a function of time at either 10 microM or 1 mM initial glutamine concentration. At 1 mM glutamine, glutamate and gamma-glutamylglutamate were the major products formed at the end of a 5-min incubation period; glutamate accounted for 46% while gamma-glutamylglutamate accounted for 33% of the glutamine utilized. With time, glutamate continued to accumulate while gamma-glutamyl peptide formation leveled off. The role of gamma-glutamyl transpeptidase was assessed by using hippurate, a physiological activator of gamma-glutamyl transpeptidase and acivicin, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, an inhibitor of gamma-glutamyl transpeptidase. Hippurate, 4 mM, increased the utilization of glutamine and the formation of glutamate, gamma-glutamyl peptides and ammonia. Exposure of cells to acivicin resulted in 98% inhibition of gamma-glutamyl transpeptidase without effecting phosphate-dependent glutaminase activity. Acivicin inhibition resulted in a decreased utilization of glutamine and product formation as compared to control; 5-oxoproline appearance fell 70%. The fractional distribution of glutamine carbon and nitrogen into its metabolic products in control, hippurate and acivicin-treated cells showed no change at the end of 60 min. The data provide evidence that gamma-glutamyl transpeptidase utilizes glutamine and forms gamma-glutamyl peptides in cultured kidney cells.     

Characterization and inhibitor sensitivity of human sperm phospholipase A2: evidence against pivotal involvement of phospholipase A2 in the acrosome reaction

The kinetic properties and inhibitor sensitivity of human sperm phospholipase A2 (PLA2; EC 3.1.1.4) were studied. Phospholipase activity was isolated from human spermatozoa by acid extraction. Hydrolysis of dipalmitoyl phosphatidylcholine was specific to the sn-2 position. Activity was sensitive to product inhibition (60% inhibition by 0.1 mM lysophosphatidylcholine). The effects of Ca2+ and sodium deoxycholate on enzyme activity were biphasic; maximal activities were observed at 0.5 mM concentration of each agent. PLA2 was stimulated (135%) by 3% dimethylsulfoxide and was inhibited by elevated ionic strength (approximately 70% inhibition with either 0.2 M NaCl or 0.2 M KCl). Two molecular forms of PLA2 were kinetically distinguishable, one with an apparent Michaelis constant and maximal reaction velocity of 3.0 microM and 0.64 mlU/mg protein and the other with respective constants of 630 microM and 32.0 mlU/mg protein. Both forms of the enzyme were Ca2+ dependent and heat stable; however, the low-Km activity was less resistant to 60 degrees C preincubation at pH 7.5 (28% inactivation of low-Km activity after 45 min, as compared to no effect on high-Km activity). Quinacrine was a noncompetitive PLA2 inhibitor with Kis for low- and high-Km activities of 0.42 mM and 0.49 mM, respectively. Trifluoperazine (calmodulin antagonist) inhibited the high-Km activity noncompetitively (Ki = 87 microM) and the low-Km activity by a mechanism consistent with the removal of a nonessential activator. Dissociation and rate constants for inactivation of low- and high-Km activities by p-bromophenacyl bromide were 0.28 mM and 0.032 min-1, and 0.73 mM and 0.066 min-1, respectively. PLA2 was inhibited by p-nitrophenyl-p'-guanidinobenzoate, at higher concentrations (10(-4)-10(-3) M) than required to inhibit trypsinlike proteinases; p-aminobenzamidine, another potent trypsin/acrosin inhibitor, stimulated (approximately 40%) PLA2 at concentrations from 2-5 mM but inhibited PLA2 (40-50%) at a concentration of 10 mM. MnCl2 (5mM) inhibited low- and high-Km PLA2 activities by 77% and 76%, respectively. Quinacrine (0.4 mM), trifluoperazine (20 microM), p-bromophenacyl bromide (20 microM), and MnCl2 (5 mM) were tested as inhibitors of the ionophore A23187-induced human acrosome reaction. Inhibition was noted only with quinacrine (32%) and MnCl2 (93%). The effect of MnCl2 was restricted to an interaction with A23187, rather than with PLA2; p-Bromophenacyl bromide inhibited (P less than 0.05) PLA2 (29%) when added to intact spermatozoa but had no effect on the acrosome reaction. PLA2 inhibition was poorly correlated with the acrosome reaction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Kinetic,ESR, and trapping evidence for in vivo binding of Mn(II) to glutamine synthetase in brain cells

Mn(II) has been proposed as a potential modulator of various important CNS enzymes, particularly glutamine synthetase, which is compartmentalized in the cytoplasm of glia. Previous studies demonstrated that total glial Mn(II) was 50–57 M, of which 30–40% occurs in the cytoplasm. In the present study, electron spin resonance (ESR) was used to determine that the concentration of free cytoplasmic Mn(II) in cultured chick glial cells is 0.8 (±0.2) M, very near K_d for the GS-Mn(II) complex. No free Mn(II) could be detected in glial mitochondria. Association of Mn(II) with brain glutamine synthetase (GS) was assessed under in vivo conditions in the presence of millimolar Mg(II) by trapping bound⁵⁴Mn(II) ions in the active site with irreversible inhibitors, namely methionine-sulfoximine (MSOX) or specific analogues thereof plus ATP. Ovine brain tissue was lysed directly into buffer containing Mn(II), 3 mM Mg(II), 1 mM MSOX, 1 mM ATP, 200 mM KCl, and 20 mM NaCl. Alternatively, primary cultures of chick glial cells were permeabilized into these inactivation mixtures. -Methyl-d,l-prothionine-S,R-sulfoximine was used to specifically inhibit the mechanistically-related enzyme -glutamyl-cysteine synthetase prior to specific inactivation of GS by -ethyl-d,l-methionine-S,R-sulfoximine. Even inthe presence of 2–3 mM Mg(II), with only 5–10 M Mn(II) present, approximately 20–30% of GS subunits were trapped with bound Mn(II). These results indicate that brain GS exhibits a high degree of specificity for binding Mn(II) over Mg(II) and that Mn(II) binds to GS to a significant extent under in vivo conditions.     

Regulation of glutamine synthetase activity in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 by the nitrogen source: effect of ammonium.

Glutamine synthetase activity from Synechocystis sp. strain PCC 6803 is regulated as a function of the nitrogen source available in the medium. Addition of 0.25 mM NH4Cl to nitrate-grown cells promotes a clear short-term inactivation of glutamine synthetase, whose enzyme activity decreases to 5 to 10% of the initial value in 25 min. The intracellular levels of glutamine, determined under various conditions, taken together with the results obtained with azaserine (an inhibitor of transamidases), rule out the possibility that glutamine per se is responsible for glutamine synthetase inactivation. Nitrogen starvation attenuates the ammonium-mediated glutamine synthetase inactivation, indicating that glutamine synthetase regulation is modulated through the internal balance between carbon-nitrogen compounds and carbon compounds. The parallelism observed between the glutamine synthetase activity and the internal concentration of alpha-ketoglutarate suggests that this metabolite could play a role as a positive effector of glutamine synthetase activity in Synechocystis sp. Despite the similarities of this physiological system to that described for enterobacteria, the lack of in vivo 32P labeling of glutamine synthetase during the inactivation process excludes the existence of an adenylylation-deadenylylation system in this cyanobacterium.     

Enzymatic basis for the calcium-induced decrease of membrane protein methyl esterification in intact erythrocytes. Evidence for an impairment of S-adenosylmethionine synthesis

The effect of Ca2+ loading, induced by the ionophore A23187, on methyl esterification of membrane proteins (i.e. bands 2.1, 3, 4.1 and 4.5) has been investigated in intact human erythrocytes. When the cells were incubated with L-[methyl-3H]methionine, 40 microM CaCl2 and 10 microM A23187 induce a 50% inhibition of membrane protein methyl esterification. This effect is selectively due to the increased intracellular Ca2+ concentration, as it is antagonized by 10 mM EGTA, and other divalent cations such as Mn2+ do not exert any inhibition. In order to clarify the mechanism(s) of the reported inhibition, the various events involved in the methyl esterification process in vivo were analyzed. L-Methionine uptake as well as protein methylase II activity are not directly affected by altered intracellular Ca2+ concentrations. Conversely in the Ca2+-loaded erythrocytes the conversion of [3H]methionine into [3H]AdoMet, catalyzed by AdoMet synthetase, decreases up to 25%. When the undialyzed erythrocyte cytosolic fraction is assayed in vitro for AdoMet synthetase the activity of the enzyme from the CaCl2/A23187-treated erythrocytes is significantly lower than the control, up to 5 mM ATP. This result suggests that in the Ca2+-loaded erythrocytes the ATP intracellular concentration is significantly lowered. The direct evaluation of ATP intracellular concentration, by HPLC, confirms a significant drop of ATP level, as a consequence of the Ca2+ loading. The removal of Ca2+ from the cells quantitatively restores both the AdoMet synthesis and the methyl esterification levels. The possible role of altered ATP intracellular concentrations as a regulatory factor in the AdoMet-dependent reactions as well as in post-translational protein methylation related to the ageing process is also discussed.     

Phytohormonal regulation of S-adenosylmethionine synthetase and S-adenosylmethionine levels in dwarf pea epicotyls.

A significant stimulation (2- to 2.5-fold) of AdoMet synthetase was witnessed in glibberellicd acid (GA3, 1 microM)-treated epicotyls of the dwarf pea (Pisum sativum). This was accompanied by a 2.4-fold increase in the endogenous pool of S-adenosylmethionine. Both abscisic acid (10 microM) and cycloheximide (20 micrograms/ml) inhibited the GA3-mediated enhancement of AdoMet synthetase activity. Three isozymes of AdoMet synthetase were detected in GA3-treated epicotyls, whereas a single activity peak was observed in controls. Thus, GA3 seems to control the induction of two new isozymes of AdoMet synthetase in the dwarf pea. By contrast, the tall pea exhibited three isozymes of AdoMet synthetase even in the absence of GA3 treatment. High concentration of L-methionine (2 mM) mimicked the GA3-elicited induction of two new isozymes of AdoMet synthetase in dwarf pea epicotyls.     

The involvement of carnitine intermediates in peroxisomal fatty acid oxidation: a study with 2-bromofatty acids

Metabolism-dependent inactivators of 3-ketothiolase I and carnitine acyltransferase I (CAT I) have been used to study the oxidation of fatty acids in intact hepatocytes. 2-Bromooctanoate inactivates mitochondrial and peroxisomal 3-ketothiolases I in a time-dependent manner. During the first 5 min of incubation, inactivation of 3-ketothiolase in mitochondria is five times faster than its inactivation in peroxisomes. Almost complete inactivation of 3-ketothiolase I in both types of organelle is achieved after incubation with 1 mM 2-bromooctanoate for 40 min. The inactivation is not affected by preincubating hepatocytes with 20 microM tetradecylglycidate (TDGA), an inactivator of CAT I, under conditions which cause greater than 95% inactivation of CAT I. 2-Bromododecanoate (1 mM) causes 60% inactivation of mitochondrial and peroxisomal 3-ketothiolases I in 40 min. These inactivations are greatly reduced by preincubating hepatocytes with 20 microM TDGA, demonstrating that 2-bromododecanoate enters both mitochondria and peroxisomes via its carnitine ester. 2-Bromopalmitate (1 mM) causes less than 5% inactivation of mitochondrial and peroxisomal 3-ketothiolases I in 40 min, but causes 95% inactivation of CAT I during this time. Incubation of hepatocytes with 10-200 microM 2-bromopalmitoyl-L-carnitine causes inactivation of mitochondrial and peroxisomal 3-ketothiolases I at similar rates. This inactivation is decreased by palmitoyl-D-carnitine during the first 5 min of incubation. Pretreating hepatocytes with 20 microM TDGA does not affect the inactivation of mitochondrial or peroxisomal 3-ketothiolase I by 2-bromopalmitoyl-L-carnitine. These results demonstrate that in intact hepatocytes, peroxisomes oxidize fatty acids of medium-chain length by a carnitine-independent mechanism, whereas they oxidize long-chain fatty acids by a carnitine-dependent mechanism.     

Characterization of leukotriene C4 synthetase in mouse peritoneal exudate cells

Cell lysates of mouse peritoneal macrophages, in the presence of reduced glutathione, converted leukotriene LTA4 to LTC4, and neither LTD4 nor LTE4 was detected. Therefore, like cultured rat basophilic leukemia cells (RBL cells), the peritoneal macrophage contains LTC4 synthetase and appears to contain little, if any, gamma-glutamyl transpeptidase. When LTA4 was added to subcellular fractions of mouse macrophage lysate, the highest specific activity of LTC4 synthetase (nmol LTC4/mg protein per 10 min) was associated with the particulate or membrane fractions (i.e., 10(4) and 10(5) X g pellets). The 10(5) X g supernatant contains approx. 1% of the specific activity and 6% of the total LTC4 synthetase activity compared with that of the 10(5) X g pellet. Conversely, the 10(5) X g supernatant had four-times more specific activity and 19-times more total GSH S-transferase activity than did the 10(5) X g pellet when evaluated using 1-chloro-2,4-dinitrobenzene (DNCB) as the substrate. LTA4 was converted to LTC4 by the membrane enzyme LTC4 synthetase in a dose-dependent manner at low LTA4 concentrations (3-50 microM) and reached a plateau of approx. 30 microM LTA4 using the macrophage 10(5) X g pellet as an enzyme source. The apparent Km value of LTC4 synthetase for LTA4 was estimated to be 5 microM based on Lineweaver-Burk plots. Enzyme in the 10(5) X g supernatant produced negligible quantities of LTC4 (1% or less of the particulate fractions) over a wide range of LTA4 concentrations. However, an enzyme in the 10(5) X g supernatant fraction presumed to be GSH S-transferase effectively catalyzes the conjugation of glutathione (GSH) with the aromatic compound DNCB. The apparent Km value of GSH S-transferase for DNCB was estimated to be 1.0-1.5 mM. On the other hand, enzyme from the membrane fraction (i.e., 10(5) X g pellet) catalyzed this reaction at a negligible rate over a wide range of DNCB concentrations. The apparent Km value of LTC4 synthetase for GSH was estimated to be 0.36 mM and the corresponding Km value estimated for the glutathione S-transferase was 0.25-0.76 mM. These values indicate similar kinetics for GSH utilization by both enzymes. These Km values are also significantly lower than the intracellular GSH levels of 2 to 5 mM. Therefore, it is suggested that the substrate limiting LTC4 synthetase activity is LTA4 and not GSH. Our results indicate that LTC4 synthetase from mouse peritoneal macrophages is a particulate or membrane-bound enzyme, as was reported by Bach et al.(ABSTRACT TRUNCATED AT 400 WORDS)     

The varied responses of different F1-ATPases to chlorpromazine

The effects of chlorpromazine on various properties of the F1-ATPases from bovine heart mitochondria (MF1), the plasma membranes of Escherichia coli (EF1), and plasma membranes of the thermophilic bacterium PS3 (TF1) have been examined. While chlorpromazine inhibited MF1 with an I0.5 of about 50 microM and EF1 with an I0.5 of about 150 microM at 23 degrees C, the ATPase activity of TF1 was stimulated by chlorpromazine concentrations up to 0.6 mM at this temperature. Maximal activation of about 20% was observed at 0.2 mM chlorpromazine at 23 degrees C. Chlorpromazine concentrations greater than 0.6 mM inhibited TF1 at 23 degrees C. At 37 degrees C the ATPase activity of TF1 was doubled in the presence of 0.5 mM chlorpromazine, the concentration at which maximal stimulation was observed at this temperature. Chlorpromazine inhibited the rate of inactivation of EF1 by dicyclohexylcarbodiimide (DCCD) at 23 degrees C and pH 6.5. Concentrations of chlorpromazine which inhibited the ATPase activity of TF1 at pH 7.0 accelerated the rate of inactivation of the enzyme by DCCD at pH 6.5, while lower concentrations of the phenothiazine, which stimulated the ATPase, had no effect on DCCD inactivation. Chlorpromazine concentrations up to 1.0 mM had no effect on the rate of inactivation of TF1 by DCCD at 37 degrees C and pH 6.5. Chlorpromazine at 0.5 mM accelerated the rate of inactivation of MF1 by 5'-p-fluorosulfonylbenzoyladenosine (FSBA), while it slowed the rate of inactivation of EF1 by FSBA. The inactivation of TF1 by FSBA in the absence of chlorpromazine was complex and was not included in this comparison. Chlorpromazine protected MF1 and EF1 against cold inactivation. Whereas 100 microM chlorpromazine afforded about 90% stabilization of MF1 at 4 degrees C, only about 30% stabilization of EF1 was observed under the same conditions in the presence of 400 microM chlorpromazine. Each of the ATPases was inactivated by the structural analog of chlorpromazine, quinacrine mustard. Whereas 5 mM ATP and 5 mM ADP protected MF1 and TF1 against inactivation by 0.5 mM quinacrine mustard, the rate of inactivation of EF1 by quinacrine mustard was accelerated fourfold by 5 mM ATP and slightly accelerated by 5 mM ADP.     

Inhibition in vitro of lipogenic enzymes from bovine (Bos taurus) mammary tissue by methylmalonyl-coenzyme A and coenzyme A

Bovine mammary fatty acid synthetase was inhibited by approximately 50% by 40 microM methylmalonyl-CoA; this inhibition was competitive with respect to malonyl-CoA (apparent Ki = 11 microM). Similarly, 6.25 microM coenzyme A inhibited the synthetase by 35% and this inhibition was again competitive (apparent Ki = 1.7 microM). Apparent Km for malonyl-CoA was 29 microM. The short-chain dicarboxylic acids malonic, methylmalonic and ethylmalonic at high concentrations (160-320 microM) and ATP (5 mM) enhanced the synthetase activity by about 50% respectively; the activating effects of methylmalonic acid and ATP on the synthetase were additive. Methylmalonyl-CoA at 50 microM concentration inhibited the partially purified acetyl-CoA carboxylase uncompetitively by 10% and the propionyl-CoA carboxylase activity of the enzyme preparation competitively (apparent Ki = 21 microM) by 40%. Malonyl-CoA also inhibited the acetyl-CoA carboxylase activity competitively (apparent Ki = 7 microM) by 35% and the propionyl-CoA carboxylating activity of the preparation competitively (apparent Ki = 4 microM) by 82%. The possibility that methylmalonyl-CoA may be a causal factor in the aetiology of the low milk-fat syndrome in high yielding dairy cows is discussed.     

Modification of sodium inactivation in myelinated nerve by Anemonia toxin II and iodate. Analysis of current fluctuations and current relaxations

(1) Na+ currents and Na+ current fluctuations were measured in single myelinated nerve fibres of Rana esculenta under voltage-clamp conditions. The process of Na+ inactivation was modified by external treatment with 7 microM Anemonia Toxin II or by internal application of 20 or 40 mM IO3(-). (2) At depolarization of 24 and 32 mV the spectral density of Na+ current fluctuations could be described as the sum of two contributions, Sh(f) and Sm(f), representing the spectrum from fluctuations of the inactivation (h) and activation (m) gates, respectively. At higher depolarizations of 40 and 48 mV the low frequency (h) fluctuations could be better fitted by the sum, Sh1(f)+Sh2(f), of two separate Lorentzian functions. (3) The Na+ current and the variance of Na+ current fluctuations between 150 and 450 ms after depolarization are increased by one order of magnitude after application of Anemonia Toxin II or IO3(-). (4) The kinetics of Na+ current inactivation were described as A1 x exp(-t/tau h1) + A2 x exp(-t/tau h2) + B. The constant, tau h1, of fast Na+ inactivation was the same in normal and modified nerve fibres. The slow inactivation time constant, tau h2, increased with increasing depolarizations in modified fibres but decreased under control conditions. In all cases tau h2 showed a similar voltage dependence as the time constant found by fitting the low frequency fluctuations of Na+ current with one Lorentzian function, Sh(f). (5) It is concluded that Anemonia Toxin II and IO3(-) modify a fraction of Na+ channels in an all-or-none manner. A lower limit of the number of modified Na+ channels is estimated from the Na+ current and the variance Na+ current fluctuations. 7 microM external Anemonia Toxin II modifies more than 17% and 20 or 40 mM internal IO3(-) more than 8% of all Na+ channels. The inactivation gates in modified channels experience an electric field different from that in normal fibres.     

Inactivation of bovine kidney cytosolic protamine kinase by the catalytic subunit of protein phosphatase 2A

Incubation of highly purified preparations of the bovine kidney cytosolic protamine kinase in the presence of near homogeneous preparations of the catalytic subunit of protein phosphatase 2A (PrP2Ac) from bovine kidney resulted in time-dependent inactivation of the protamine kinase. By contrast, incubation of bovine kidney cytosolic casein kinase II with PrP2Ac had no effect on the activity of this casein kinase II. In the presence of 10 mM sodium fluoride, 10 mM inorganic orthophosphate, 1 mM pyrophosphate or 0.1 mM ATP, the inactivation of the protamine kinase by PrP2Ac was completely inhibited. Half-maximal inhibition by ATP occurred at about 20 microM. The rate of inactivation of the protamine kinase by PrP2Ac was unaffected by Mg2+, Mn2+, Ca2+, EDTA or EGTA at 1 mM. The results strongly indicate that the activity of the cytosolic protamine kinase is regulated by phosphorylation/dephosphorylation.     

Cytoprotective effect of copper(II) complexes against ethanol-induced damage to rat gastric mucosa.

The cytoprotective effect of various copper(II) complexes on the gastric mucosa damage induced by acute intragastric administration of ethanol was investigated. For in vitro experiments, the following copper(II) complexes were tested: Cu(II)(L-Trp)(L-Phe), Cu(II)(L-Leu)Cu(II)(L-Leu-Leu)(L-Leu), Cu(II)(L-Phe-L-Leu), Cu(II)(Gly-His-Lys), and Cu(II)(cyHis)2(ClO4)2. Inorganic copper such as CuSO4 was also tested. The free radical generating system, acting for 2 hr on cardial and fundic mucosa scrapings or mucosal microsomes, was Fe++ (20 microM)/ascorbate (0.25 mM). We found a marked inhibition to 75% of lipid peroxidation in the range 10-100 mM, regardless of whether copper was given in complexed or inorganic form. The results suggest that nontoxic copper(II)-amino acid complexes are able to neutralize oxygen-derived free radicals. In addition, copper(II) complexes suppressed membrane lipid peroxidation when mucosa homogenates were exposed to t-butyl hydroperoxide (1-20 microM) plus Fe++ (50 microM). In vivo experiments on rat stomachs, pretreated p.o. by gavage either with Cu(II)(L-Trp)(L-Phe) as paradigmatic agent or with copper sulphate at equivalent doses in the range 3-30 mg/kg body weight showed a significant decrease (30 min after 95% ethanol administration) in the number and severity of mucosal hemorrhagic lesions. In the gastric mucosa scrapings of copper-treated rats after ethanol exposure, we found that malondialdehyde and conjugated diene levels were unchanged compared to those of untreated controls; five enzyme activities released from lysosomes were near control values. In isolated mucosal cells, whether or not pretreated with 200 microM solution of either Cu(II)(L-Trp)(L-Phe) or CuSO4, the release of cathepsin D activity was also unmodified. The results suggest that the cytoprotective effect of Cu(II) complexes against ethanol-induced mucosal lesions was not associated in vivo to lipid peroxidation.     

Purification and characterization of two Cl- -activated aminopeptidases hydrolysing basic termini from human skeletal muscle

Two aminopeptidases (I and II), hydrolysing basic termini, were purified to homogeneity (as judged by polyacrylamide gel electrophoresis) from human quadriceps muscle by anion-exchange chromatography and preparative electrophoresis. The electrophoretic migration rate of II was approximately 80% of that of I. Both enzymes had the following properties: optimum activity was at pH 6.5; addition of 0.15 M Cl- or Br- anions resulted in a 20-fold or 10-fold increase in activity respectively. There was little or no increase in activity on the addition of other anions, or divalent cations (0.05-5mM). Approximately 50% inhibition of activity was obtained in the presence of bestatin (0.1 microM), rho-hydroxymercuriphenylsulphonic acid (0.1 microM), EDTA (10 mM), 1,10-phenanthroline (100 microM), N-ethylmaleimide (1 mM) and But-Thr-Phe-Pro (0.5 mM). The molecular mass was 72 000 Da (gel filtration). Only the arginyl and lysyl 7-amino-4-methylcoumarin (Amc) derivatives were appreciably hydrolysed; approximate Km values for the reaction of I and II with these substrates (10-250 microM) were estimated as follows: Arg-Amc, KmI = 70 microM, KmII = 270 microM; Lys-Amc KmI = 280 microM, KmII = 400 microM. Both enzymes hydrolysed dipeptides with Arg or Lys as the NH2-terminal amino acid, however this was not an absolute requirement for dipeptide hydrolysis. The action of I and II on physiologically active oligopeptides was very restricted, with only bradykinin, proangiotensin and neurotensin being appreciably degraded. The breakdown of these peptides did not occur by classical aminopeptidase action (i.e. hydrolysis of the NH2-terminal residues), but via cleavage of internal peptide bonds. These results suggest that I and II may be isoenzymes of a Cl- -requiring, thiol-type aminopeptidase, which hydrolyses basic termini. These enzymes may act primarily as dipeptidases, with a very restricted mode of action in the degradation of naturally occurring oligopeptides.     

Proteolysis as a probe of ligand-associated conformational changes in rat carbamyl phosphate synthetase I

Elastase, V8 protease, subtilisin, trypsin, and chymotrypsin all cleaved the 1462-residue polypeptide of rat carbamyl phosphate synthetase I in segment C 160-180 residues from the COOH-end. Its activator N-acetylglutamate (AcGlu) increased the rate of cleavage approximately ninefold, presumably by binding preferentially to the conformation in which C is exposed. ATP/Mg2+ prevented proteolysis both +/- AcGlu. Kd,app for AcGlu (66 microM) and ATP (4.2 microM with AcGlu and 5 mM Mg2+) was estimated from the pseudo-first-order rate constants for inactivation caused by cleavage with elastase at C. Chymotrypsin and trypsin also hydrolyzed the enzyme, independent of AcGlu, at site D within less than 20 residues of the COOH-end. D was protected by ATP only in the presence of AcGlu and K+, and enzyme hydrolyzed exclusively at D had greater than 30-fold higher Km's for AcGlu and ATP. Digestion by trypsin at a third site (B) approximately 530 residues upstream from C appeared to occur subsequent to hydrolysis at C. Slow cleavage by elastase at an additional site (A) to give 360- and 1100-residue peptides was unaffected by AcGlu and ATP, and caused only modest loss of activity. These peptides were isolated by chromatography on DEAE-cellulose. Assignment of the smaller one to the NH2-end on the basis of its cysteine content places site A in the junction between the segments homologous to the small glutaminase and large synthetase subunits of Escherichia coli carbamyl phosphate synthetase II. Neither peptide alone was active; maximal regain of activity (approximately 25%) occurred on combining them in equimolar proportions. The sizes of the peptides produced by further digestion of the site A digest gave the approximate locations of the other sites. Sites A (Ala-417) and B (Arg-787) have recently been identified by NH2-terminal sequencing (S. G. Powers-Lee and K. Corina (1986) J. Biol. Chem. 261, 15349-15352). Reasons for the low value of KAcGlu,app are examined, and protection by ATP is discussed in relation to previous models for the conformational equilibria of the enzyme.     

The inhibitory effect of the fungicides captan and captafol on eukaryotic topoisomerases in vitro and lack of recombinagenic activity in the wing spot test of Drosophila melanogaster

In studies on the mechanisms of mutagenic and carcinogenic action of captan and captafol-related chloroalkylthiocarboximide fungicides, two effects were tested: (i) the effect of both compounds on the activity of eukaryotic topoisomerases I and II in vitro, and (ii) their mutagenic and recombinagenic activity in the somatic mutation and recombination test (SMART) in wing cells of Drosophila melanogaster. Only captafol inhibited the activity of topoisomerase I (10-20% inhibition of activity in the range of 10-100microM). In contrast, both chemicals decreased the activity of topoisomerase II already at 1microM concentration (50 and 20% inhibition of activity by captafol and captan, respectively).Genotoxicity was tested in vivo by administrating both compounds by acute (3h) and chronic feeding (48h) of 3-day-old larvae. In acute feeding, captan and captafol demonstrated positive results only for small single and total spots in 10-100mM exposure concentration range. Both chemicals were inconclusive for large single spots, as well as for twin spots. In chronic treatment, captan showed positive results only for small single and total spots at 2.5 and 5mM concentrations. Captafol gave inconclusive results over all concentrations tested. The results of the acute treatment experiments which have been performed at very high doses (50% toxicity at higher doses) indicate very weak overall mutagenic activity of both test fungicides.     

Regulation of the activity of the Bacillus licheniformis A5 glutamine synthetase

The regulation of glutamine synthetase activity by positive and negative effectors of enzyme activity singularly and in combinations was studied by using a homogeneous enzyme preparation from Bacillus licheniformis A5. Phosphorylribosyl pyrophosphate at concentrations greater than 2mM stimulated glutamine synthetase activity by approximately 70%. The concentration of phosphorylribosyl pyrophosphate required for half-maximal stimulation of enzyme activity was 0.4 mM. Results obtained from studies of fractional inhibition of glutamine synthetase activity were consistent with the presence of one allosteric site for glutamine binding (apparent I0.5, 2.2mM) per active enzyme unit at a glutamate concentration of 50 mM. At a glutamate concentration of 30 mM or less, the data were consistent with the enzyme containing two binding sites for glutamine (one of which was an allosteric site with an apparent I0.5 of 0.4 mM). Bases on an analysis of the response of glutamine synthetase activity to positive and negative effectors in vitro and to the intracellular concentration of these effectors in vivo, the primary modulators of glutamine synthetase activity in B. licheniformis A5 appear to be glutamine and alanine (apparent I0.5, 5.2mM).