Non-Enzymatic Glycosylation (or Glycation) and Inhibition of the Pig Heart Cytosolic Aspartate Aminotransferase by Glyceraldehyde 3-Phosphate

Glyceraldehyde 3-phosphate (Glyc3P), a glycolytic intermediate, non-enzymatically glycosylated (or glycated) and inhibited the pig heart cytoplasmic aspartate aminotransferase (cAAT). Glyc3P (5.0 mM) decreased cAAT activity by 47% after 1 min at 23 degrees C. cAAT activity remained unchanged after a 24 h incubation with either glucose 6-phosphate (5.0 mM) or ribose 5-phosphate (5.0 mM). Increasing the incubation pH from 6.4 to 7.8 or the incubation temperature from 23 degrees C to 50 degrees C enhanced Glyc3P's inhibitory effect on cAAT activity. Glyc3P (250-500 μM) decreased the thermal stability of cAAT as evidenced by lowering the T(m) or temperature that caused a 50% irreversible loss of cAAT activity (69 degrees C, control; 58.5 degrees C, 500 μM Glyc3P). Glyc3P decreased cAAT amino group content and increased glycation products, which were measured by adduct formation, fluorescence and protein crosslinking.     

Glycation of aspartate aminotransferase and conformational flexibility

Glycation of proteins alters biological function and changes cellular processes. Our study investigated the conformational changes that accompany glycation using the cardiac aspartate aminotransferase (cAAT). We examined the effects of brief and prolonged exposure of cAAT to glyceraldehyde (Glyc) and ribose 5-phosphate (R5P). When cAAT was briefly incubated (3.5 h) with Glyc (500 microM) or R5P (5 mM) at 37 degrees C, cAAT activity and 1-anilinonaphthalene 8-sulfonate (ANS) binding increased relative to control. After prolonged incubation (64 h) with Glyc (500 microM) or R5P (5 mM) at 37 degrees C, activity and ANS binding decreased relative to control. Furthermore, upon prolonged incubation of cAAT with 500 microM Glyc (14.5 h) or 2 mM R5P (64.25 h) at 37 degrees C, the denaturation curves shifted to the right relative to control. We conclude that upon brief incubation with Glyc and R5P, cAAT exhibited a more open and flexible structure and upon prolonged incubation, a more rigid structure.     

Carnosine prevents the glycation-induced changes in electrophoretic mobility of aspartate aminotransferase

Carbohydrate-derived aldehydes cause irreversible loss of protein function via glycation. We previously observed that glyceraldehyde 3-phosphate (Glyc3P) abolishes the enzyme activity of cardiac aspartate aminotransferase (cAAT). We also examined the protective effects of carnosine against Glyc3P-induced loss of enzyme activity. The present study looked at carnosine's prevention of Glyc3P-induced change in protein structure. Purified cAAT (2 mg protein/mL) was incubated with various concentrations of carnosine (1-20 mM) in the presence of Glyc3P (500 microM) for 4 days at 37 degrees C. Following incubation, samples were analyzed by SDS-polyacrylamide gel electrophoresis. Carnosine showed prevention of protein modification at carnosine-to-Glyc3P ratios of 10:1 or greater. There was a progressive loss of the unmodified cAAT protein band as Glyc3P concentration was increased. Additionally, the gel position of the Glyc3P-modified cAAT protein varied over time. The apparent molecular weight (MWapp) of the Glyc3P-modified cAAT protein that formed after 1 day at 37 degrees C (500 microM) was greater than its MWapp after 2 days, suggesting that a chemical rearrangement of the initial adduct occurs. These observations support the hypothesis that carnosine is an antiglycation agent and that its mechanism of action involves prevention of protein modification.     

Carnosine promotes the heat denaturation of glycated protein

Glycation alters protein structure and decreases biological activity. Glycated proteins, which accumulate in affected tissue, are reliable markers of disease. Carnosine, which prevents glycation, may also play a role in the disposal of glycated protein. Carnosinylation tags glycated proteins for cell removal. Since thermostability determines cell turnover of proteins, the present study examined carnosine's effect on thermal denaturation of glycated protein using cytosolic aspartate aminotransferase (cAAT). Glycated cAAT (500 microM glyceraldehyde for 72h at 37 degrees C) increased the T(0.5) (temperature at which 50% denaturation occurs) and the Gibbs free energy barrier (DeltaG) for denaturation. The enthalpy of denaturation (DeltaH) for glycated cAAT was also higher than that for unmodified cAAT, suggesting that glycation changes the water accessible surface. Carnosine enhanced the thermal unfolding of glycated cAAT as evidenced by a decreased T(0.5) and a lowered Gibbs free energy barrier. Additionally, carnosine decreased the enthalpy of denaturation, suggesting that carnosine may promote hydration during heat denaturation of glycated protein.     

Inhibition of aspartate aminotransferase by glycation in vitro under various conditions

Incubation of 50 mM D-glucose with aspartate aminotransferase (AST, EC 2.6.1.1) preparations (purified pig heart enzyme or a rat liver 20,000 x g supernatant) at 25 degrees C had no effect on enzyme activity. 50 mM D-fructose or D-ribose gradually inhibited pig heart AST under the same conditions to zero activity after 14 days. 50 mM DL-glyceraldehyde decreased enzyme activity to zero after 6 days of incubation. The inhibition of pig heart AST by 50 mM D-fructose or D-ribose was marked even at a temperature of 4 degrees C but it was less pronounced than at 25 degrees C. There was no effect of 0.5 mM 2-oxoglutarate on AST activity during incubation, while the presence of 25 mM L-aspartate decreased it rapidly. 0.5 mM 2-oxoglutarate partly prevented inhibition of AST by D-ribose or D-fructose, while an analogous experiment with 25 mM aspartate resulted in a rapid decline similar to that in the absence of sugars.     

Phytase activity in Cryptococcus laurentii ABO 510

Ten Cryptococcus strains were screened for phytase activity, of which the Cryptococcus laurentii ABO 510 strain showed the highest level of activity. The cell wall-associated enzyme displayed temperature and pH optima of 62 degrees C and 5.0, respectively. The enzyme was thermostable at 70 degrees C, with a loss of 40% of its original activity after 3 h. The enzyme was active on a broad range of substrates, including ATP, D-glucose 6-phosphate, D-fructose 1,6-diphosphate and p-nitrophenyl phosphate (p-NPP), but its preferred substrate was phytic acid (K(m) of 21 microM). The enzyme activity was completely inhibited by 0.5 mM inorganic phosphate or 5 mM phytic acid, and moderately inhibited in the presence of Hg(2+), Zn(2+), Cd(2+) and Ca(2+). These characteristics suggest that the Cry. laurentii ABO 510 phytase may be considered for application as an animal feed additive to assist in the hydrolysis of phytate complexes to improve the bioavailability of phosphorus in plant feedstuff.     

Effect of modification of cell calcium status on lectin activity

Effects of oryzalin (10 microM), an inhibitor of microtubule polymerization, on the activity of soluble and cell wall lectins were studied in 7 day-old seedlings of unhardened (23 degrees C) and cold acclimated (7 days at 2-3 degrees C) winter wheat (Triticum aestivum L.). Seedlings were grown in the presence of 25 microM and 1 mM Ca2+, 500 microM verapamil, 250 microM chlorpromazine or without modifiers of calcium status in the medium. Inhibitor of the microtubule polymerization inhibitor, likely as inhibitors of Ca(2+)-signal, decreased the activity of soluble lectins and increased that of cell wall lectins. Apparently, injury of microtubule phosphorylation results in a more considerable microtubule disorganization, than that observed after oryzalin effect. A low Ca2+ concentration (25 microM) depressed, while a high concentration (1 mM) prompted microtubule sensibility to oryzalin. Such an effect of high Ca2+ concentration may be related to destabilizative action of Ca(2+)-calmodulin in these conditions, because chlorpromazine decreased oryzalin-induced increase in the activity of cell wall lectins with 1 mM Ca2+. It is concluded that the activity of cell wall lectins depends on the microtubule status that is regulated by calcium signal.     

Short-term feedback inhibition of photosynthesis in wheat leaves supplied with sucrose and glycerol at two temperatures

The inhibition of photosynthesis by reduced sink demand or low rates of end product synthesis was investigated by supplying detached wheat (Triticum aestivum L. cv. Tauro) leaves with 50 mM sucrose, 50 mM glycerol or water through the transpiration stream for 2 h, either at 23 or 12 °C. Lowering the temperature and sucrose and glycerol feeding decreased photosynthetic oxygen evolution at high irradiance and saturating CO₂. The decrease in temperature reduced the pools of sucrose and starch, and the ratio glucose 6-phosphate (G6P)/fructose 6-phosphate (F6P), while it increased the concentrations of G6P and F6P (hexose phosphates). Sucrose feeding, in contrast to glycerol feeding, increased sucrose, glucose and fructose contents and the G6P/F6P ratio. Sucrose and glycerol incubations at 23 °C, as well as decreasing the temperature in leaves incubated in water, increased the concentration of triose-phosphates (glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, TP) and decreased the glycerate 3-phosphate (PGA) content, thus increasing the TP/PGA ratio; they also tended to increase the ribulose 1,5-bisphosphate (RuBP) content and the RuBP/PGA ratio. Sucrose and glycerol feeding at 12 °C and the decrease in temperature of leaves incubated in these solutions decreased TP and RuBP contents and the TP/PGA and RuBP/PGA ratios. The results suggest that the phosphate limitation caused by accumulation of end products, restriction of their synthesis and sequestration of cytosolic phosphate can inhibit photosynthesis through decreased carboxylation of RuBP or, with increased phosphate limitation, through lowered supply of ATP. This revised version was published online in August 2006 with corrections to the Cover Date.     

Suppression of the accumulation of triosephosphates and increased formation of methylglyoxal in human red blood cells during hyperglycaemia by thiamine in vitro

The accumulation of triosephosphates and the increased formation of the potent glycating agent methylglyoxal in intracellular hyperglycaemia are implicated in the development of diabetic complications. A strategy to counter this is to stimulate the anaerobic pentosephosphate pathway of glycolysis by maximizing transketolase activity by thiamine supplementation, with the consequent consumption of glyceraldehyde-3-phosphate and increased formation of ribose-5-phosphate. To assess the effect of thiamine supplementation on the accumulation of triosephosphates and methylglyoxal formation in cellular hyperglycaemia, we incubated human red blood cell suspensions (50% v/v) in short-term culture with 5 mM glucose and 50 mM glucose in Krebs-Ringer phosphate buffer at 37 degrees C as models of cellular metabolism under normoglycaemic and hyperglycaemic conditions. In hyperglycaemia, there is a characteristic increase in the concentration of the triosephosphate pool of glycolytic intermediates and a consequent increase in the concentration and metabolic flux of the formation of methylglyoxal. The addition of thiamine (50-500 microM) increased the activity of transketolase, decreased the concentration of the triosephosphate pool, decreased the concentration and metabolic flux of the formation of methylglyoxal, and increased the concentration of total sedoheptulose-7-phosphate and ribose-5-phosphate. Biochemical changes implicated in the development of diabetic complications were thereby prevented. This provides a biochemical basis for high dose thiamine therapy for the prevention of diabetic complications.     

Oxidant activity in skeletal muscle fibers is influenced by temperature, CO2 level, and muscle-derived nitric oxide

Free radicals are produced continuously by skeletal muscle fibers. Extracellular release of reactive oxygen species (ROS) and nitric oxide (NO) derivatives has been demonstrated, but little is known about intracellular oxidant regulation. We used a fluorescent oxidant probe, 2',7'-dichlorofluorescin (DCFH), to assess net oxidant activity in passive muscle fiber bundles isolated from mouse diaphragm and studied in vitro. We tested the following three hypotheses. 1) Net oxidant activity is decreased by muscle cooling. 2) CO(2) exposure depresses intracellular oxidant activity. 3) Muscle-derived ROS and NO both contribute to overall oxidant activity. Our results indicate that DCFH oxidation was diminished by cooling muscle fibers from 37 degrees C to 23 degrees C (P < 0.001). The rate of DCFH oxidation correlated positively with CO(2) exposure (0-10%; P < 0.05) and negatively with concurrent changes in pH (7.0-8.5; P < 0.05). Separate exposures to anti-ROS enzymes (superoxide dismutase, 1 kU/ml; catalase, 1 kU/ml), a glutathione peroxidase mimetic (ebselen, 30 microM), NO synthase inhibitors (N(omega)-nitro-l-arginine methyl ester, 1 mM; N(omega)-monomethyl-l-arginine, 1 mM), or an NO scavenger (hemoglobin, 1 microM) each inhibited DCFH oxidation (P < 0.05). Oxidation was increased by hydrogen peroxide, 100 microM, an NO donor (NOC-22, 400 microM), or the substrate for NO synthase (l-arginine, 5 mM). We conclude that net oxidant activity in resting muscle fibers is 1) decreased at subphysiological temperatures, 2) increased by CO(2) exposure, and 3) influenced by muscle-derived ROS and NO derivatives to similar degrees.     

Synthesis of acetyl coenzyme A by carbon monoxide dehydrogenase complex from acetate-grown Methanosarcina thermophila.

The carbon monoxide dehydrogenase (CODH) complex from Methanosarcina thermophila catalyzed the synthesis of acetyl coenzyme A (acetyl-CoA) from CH3I, CO, and coenzyme A (CoA) at a rate of 65 nmol/min/mg at 55 degrees C. The reaction ended after 5 min with the synthesis of 52 nmol of acetyl-CoA per nmol of CODH complex. The optimum temperature for acetyl-CoA synthesis in the assay was between 55 and 60 degrees C; the rate of synthesis at 55 degrees C was not significantly different between pHs 5.5 and 8.0. The rate of acetyl-CoA synthesis was independent of CoA concentrations between 20 microM and 1 mM; however, activity was inhibited 50% with 5 mM CoA. Methylcobalamin did not substitute for CH3I in acetyl-CoA synthesis; no acetyl-CoA or propionyl coenzyme A was detected when sodium acetate or CH3CH2I replaced CH3I in the assay mixture. CO could be replaced with CO2 and titanium(III) citrate. When CO2 and 14CO were present in the assay, the specific activity of the acetyl-CoA synthesized was 87% of the specific activity of 14CO, indicating that CO was preferentially incorporated into acetyl-CoA without prior oxidation to free CO2. Greater than 100 microM potassium cyanide was required to significantly inhibit acetyl-CoA synthesis, and 500 microM was required for 50% inhibition; in contrast, oxidation of CO by the CODH complex was inhibited 50% by approximately 10 microM potassium cyanide.     

Aldehyde oxidation in human placenta. Purification and properties of 1-pyrroline-5-carboxylate dehydrogenase.

The human placenta contains a considerable amount of 1-pyrroline-5-carboxylate dehydrogenase (23 +/- 6 micrograms/g; n = 12), about 25% of the concentration present in liver. The enzyme is the only form in placenta that oxidizes short- and medium-chain aldehydes, which facilitates its purification from this organ. It can be purified to homogeneity by successive chromatographies on DEAE-cellulose, 5'-AMP-Sepharose and Sephacryl S-300. From 500 g of tissue, about 2.1 units of enzyme can be obtained with a 12% yield. Placental 1-pyrroline-5-carboxylate dehydrogenase is a dimer of Mr-63,000 subunits. It exhibits a pI of 6.80-6.65, and is specific for 1-pyrroline-5-carboxylate, the cyclic form of glutamate gamma-semialdehyde (Km = 0.17 mM, kcat. = 870 min-1), although it also oxidizes short-chain aliphatic aldehydes such as propionaldehyde (Km = 24 mM, kcat. = 500 min-1). These properties are very close to those of the liver enzyme, indicating a strong similarity between the enzyme forms from both organs. The enzyme is highly sensitive to temperature, showing 50% inhibition after incubation for 0.8 min at 45 degrees C or after 23 min at 25 degrees C. It is irreversibly inhibited by disulfiram, and a molar ratio inhibitor: enzyme of 60:1 produced 50% inhibition after incubation for 10 min. A subcellular-distribution study indicates that the enzyme is located in two compartments: the mitochondria, with 60% of the total activity, and the cytosol, with 40% activity. The physiological role of the enzyme in placental amino acid metabolism is discussed.     

Lithium-induced accumulation of inositol 1-phosphate during cholecystokinin octapeptide- and acetylcholine-stimulated phosphatidylinositol breakdown in dispersed mouse pancreas acinar cells

Dispersed mouse pancreas acinar cells were prepared in which phosphatidylinositol had been labeled with myo[2-3H]inositol. During incubation with 0.3 microM cholecystokinin octapeptide (CCK-8) for 15 min, there was a loss of [3H]phosphatidylinositol radioactivity (23%) and a 3-fold gain in trichloroacetic acid-soluble radioactivity. Replacement of NaCl by up to 58 mM LiCl did not significantly affect the amount of CCK-8-stimulated [3H]phosphatidylinositol breakdown or the gain in acid-soluble radioactivity. However, in normal medium, the product of phosphatidylinositol breakdown was almost all inositol, whereas in Li+-containing medium, the product was almost all inositol 1-phosphate. Similar results were obtained with acetylcholine which, in the presence of Li+, gave a dose-responsive increase in inositol 1-phosphate over the concentration range of 0.1 to 10 microM. No increased accumulation of [3H]inositol diphosphate or [3H]inositol triphosphate was detected in stimulated cells. Time courses in the presence of Li+ indicated that the formation of inositol 1-phosphate preceded the formation of inositol. Addition of up to 50 mM myoinositol to the incubation medium showed no diluting effect on the amount of [3H]inositol 1-phosphate found. The accumulation of inositol 1-phosphate is presumably due to the known ability of Li+ to inhibit myoinositol 1-phosphatase. The results provide clear evidence that stimulated phosphatidylinositol breakdown involves a phospholipase C type of phosphodiesterase activity. 1.25 mM Li+ gave half-maximal inositol 1-phosphate accumulation. This is close to the range of plasma Li+ levels which is used therapeutically in psychiatric disorders. In unstimulated cells, [3H]inositol 1-phosphate accumulation in the presence of Li+ corresponded to a breakdown rate for [3H]phosphatidylinositol of 2 to 3%/h.     

Phosphatidylglycerophosphate synthase activity in Saccharomyces cerevisiae

Cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDP-diacylglycerol): sn-glycerol-3-phosphate phosphatidyltransferase (phosphatidylglycerophosphate synthase, EC 2.7.8.5) activity was characterized from the mitochondrial fraction of Saccharomyces cerevisiae. The pH optimum for the reaction was 7.0. Maximum activity was dependent on manganese (0.1 mM), magnesium (0.3 mM), or cobalt (1 mM) ions and the nonionic detergent Triton X-100 (1 mM). The apparent Km values for CDP-diacylglycerol and glycerol-3-phosphate were 33 and 27 microM, respectively. Optimal activity was at 30 degrees C with an energy of activation of 5.4 kcal/mol (1 cal = 4.1868 J). Phosphatidylglycerophosphate synthase activity was thermally labile above 40 degrees C. p-Chloromecuriphenylsulfonic acid, N-ethylmaleimide, and mercurous ions inhibited activity. Phosphatidylglycerophosphate synthase activity was partially solubilized from the mitochondrial fraction with 1% Triton X-100.     

Characterization of a phosphate transport system in human fibroblast lysosomes.

The uptake of [32P]KH2PO4 by Percoll-purified human fibroblast lysosomes at pH 7.0 was investigated to determine if lysosomes contain a transport system recognizing phosphate. Lysosomal phosphate uptake was linear for the first 2 min, attained a steady state by 8-10 min at 37 degrees C, and was not Na+ or K+ dependent. Upon entering lysosomes, [32P]phosphate was rapidly metabolized to trichloroacetic acid-soluble and trichloroacetic acid-insoluble products. After 1-min incubations, 50% of the radioactivity recovered from lysosomes was in the form of inorganic phosphate; and after a 2.5-min incubation, 27% of the radioactivity was recovered as inorganic phosphate. When lysosomes are loaded with radioactivity by incubation with 0.03 mM [32P]KH2PO4 for 25 min and then washed at 4 degrees C, lysosomes fail to release the accumulated radioactivity during a subsequent incubation at 37 degrees C. Lysosomal phosphate uptake gave linear Arrhenius plots (Q10 = 1.8) and was inversely proportional to medium osmolarity. Phosphate uptake was maximal at pH 5-6, half-maximal at pH 7.1, with little transport activity at pH greater than 8, suggesting that the transport system recognizes the monobasic form of phosphate. Lysosomal phosphate uptake is saturable, displaying a Km of 5 microM at pH 7.0 and 37 degrees C. High specificity for phosphate is demonstrated since large concentrations of Na2SO4, NaHCO3, KCl, NaCl, 5'-AMP, or the anion transport inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, have no effect on lysosomal phosphate transport. In contrast, the phosphate analog, arsenate, strongly inhibits lysosomal phosphate uptake in a competitive manner with a Ki of 7 microM. Pyridoxal phosphate, CTP, adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP), and glucose 6-phosphate were found to be noncompetitive inhibitors of lysosomal phosphate uptake displaying Ki values of 80-250 microM. When lysosomes are incubated with [gamma-32P]ATP, the lysosomal membrane ATPase hydrolyzes the ATP to form inorganic phosphate which then enters lysosomes by this lysosomal phosphate transport route.     

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.     

Entrapment of Sorghum root oxalate oxidase into polyvinyl alcohol membrane

A Cl- and NO3- insensitive oxalate oxidase, purified from the roots of 10-day old seedlings of grain Sorghum has been immobilized on polyvinyl alcohol (PVA) membrane through entrapment with 96.07% retention of initial activity. The membrane bound enzyme showed an increase in optimum pH (from 5.0 to 6.5), time of incubation (from 5 to 10 min) and Km for oxalate (from 0.38 to 6.23 mM), but decrease in incubation temperature for maximum activity (from 37 to 30 degrees C) and Vmax (from 70 nmol/min/ml to 9.7 nmol H2O2/min) and was unaffected by Cl- and NO3. The membrane bound enzyme lost 50% of its initial activity after 30 days of storage at room temperature. The use of membrane bound oxalate oxidase in determination of serum oxalate of urinary stone patients is demonstrated.     

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)     

Inhibition of mitochondrial carbonic anhydrase and ureagenesis: a discrepancy examined

The amount of urea synthesized in intact guinea pig hepatocytes in 60 min ([urea]t=60), was determined at 37 degrees C in Krebs-Henseleit buffer plus (in mM) 10 NH4Cl, 5 lactate, and 10 ornithine in 5% CO2-95% O2. The concentrations of sulfonamide carbonic anhydrase (CA) inhibitors required to reduce the rate of urea synthesis by 50% (I50) were (in mM): 0.07 ethoxzolamide, 0.5 methazolamide, 0.7 acetazolamide, and 5.0 p-aminomethylbenzenesulfonamide. At 37 degrees C acetazolamide and ethoxzolamide reduced citrulline synthesis by intact mitochondria in medium containing (in mM) 50 3-(N-morpholino)propanesulfonic acid, 35 KCl, 5 KH2PO4, 2 adenosine triphosphate, 10 ornithine, 10 NH4Cl, 1 [ethylene-bis(oxyethylenenitrile)]tetraacetic acid, 1 MgCl2, 20 pyruvate, and 25 KHCO3 (pH 7.4) in 5% CO2-95% O2; the inhibition by ethoxzolamide was not decreased greater than 50%; 25% inhibition was achieved by 0.65 microM ethoxzolamide. Inhibition constant (Ki) values for CA activity of disrupted mitochondria at 37 degrees C were 0.03 microM ethoxzolamide and 0.16 microM acetazolamide, and for disrupted hepatocytes were 150 microM ethoxzolamide and 50 microM acetazolamide. p-Aminomethylaminosulfonamide-affinity column purification yields one band of 29,000 mol wt for CA V purified from disrupted mitochondria; homogenized whole-liver supernatant yields an additional band of 20,000 mol wt (at greater than 100 times the concentration of CA V), which has some glutathione S-transferase activity. It is concluded that this 20,000-mol wt protein modifies the potency of ethoxzolamide in the liver cytosol.