Difference in kinetic properties of phosphorylated intermediates formed in the forward and backward reactions of solubilized Ca2+-ATPase of sarcoplasmic reticulum

Solubilized Ca2+-ATPase (SSR) was prepared by solubilizing fragmented sarcoplasmic reticulum (FSR) with a nonionic detergent (C12E8) then displacing the detergent with Tween 80, using a DEAE-cellulose column. The kinetic properties of the phosphorylated intermediate (EP) formed by the reaction of SSR with ATP were compared with those of EP formed by the reaction with Pi. The time course of decay of E32P formed with 4 microM AT32P in the presence of 19 mM CaCl2 and 10 mM MgCl2 (forward reaction) was measured by adding 0.4 mM unlabeled ATP and 10 mM Pi at pH 6.0 and 30 degrees C. The rate of E32P decay was accelerated by 0.4 mM ADP. On the other hand, when the time course of decay of E32P formed with 10 mM 32Pi in the presence of 5 mM EGTA and 10 mM MgCl2 (backward reaction) was measured by adding 0.4 mM unlabeled ATP and 15 mM CaCl2, the rate of E32P decay was unaffected by 0.4 mM ADP. AT32P was produced on adding ADP to E32P formed with AT32P in the presence of 10 mM CaCl2 and 10 mM MgCl2, while no AT32P was produced on adding ADP to E32P formed with 32Pi in the presence of 5 mM EGTA and 10 mM MgCl2, even when 15 mM CaCl2 was added simultaneously with ADP.     

Analysis of responses of Leidynema appendiculata to acetylcholine using electrophysiological techniques

The electrophysiological responses of adult females of the insect-parasitic nematode Leidynema appendiculata, to 100 mM, 10 mM, and 1 mM acetylcholine were concentration dependent, with stimulation by 100 mM acetylcholine giving the largest increase in spike activity. By contrast, the delay in response was not concentration dependent. No increase in activity was obtained on stimulation with either 0.1 mM acetylcholine or phosphate-buffered saline. The lack of response to 0.1 mM acetylcholine was confirmed by sequential exposure of L. appendiculata to 0.1 mM acetylcholine and subsequently to 10 mM acetylcholine.     

Normalization of glucose entry under the high glucose condition by phlorizin attenuates the high glucose-induced morphological and functional changes of cultured bovine retinal pericytes

We previously reported that sodium-dependent glucose uptake is present in bovine retinal pericytes and that phlorizin normalizes its glucose consumption under high glucose conditions. To clarify the effect of phlorizin on morphological and functional change of retinal pericytes under high glucose conditions, retinal pericytes were incubated in media with 5 mM glucose, 30 mM glucose, and 30 mM glucose plus 0.2 mM phlorizin for 7 days. The diameter of cells in the concentrations of glucose more than 10 mM were significantly larger than those in 5 mM glucose and 30 mM glucose plus phlorizin. Glucose, sorbitol and fructose contents of the cells in 30 mM glucose were significantly increased compared with those in 5 mM glucose, and were normalized by phlorizin. Thymidine uptake in the concentrations of glucose more than 20 mM was significantly decreased compared with that in 5 mM glucose. Myoinositol uptake, and DNA in 30 mM glucose were significantly reduced, and were normalized with phlorizin. Myoinositol content in 30 mM glucose was the same as that in 5 mM glucose, but was significantly decreased by phlorizin. The ratios of glucose to sorbitol or fructose in 30 mM glucose were significantly decreased, compared with those in 5 mM glucose and 30 mM glucose plus phlorizin. Therefore, the cellular enlargement and decreased DNA synthesis in cultured bovine retinal pericytes with abnormal glucose metabolism under high glucose conditions are attenuated by phlorizin, independent of the cellular myoinositol content.     

Augmentation of cytostatic effect of recombinant human lymphotoxin and involvement of glutathione redox cycle.

The effect of buthionine sulfoximine (BSO), an inhibitor of glutathione biosynthesis, in combined use with a nitrosourea derivative, ACNU, on the cytostatic effect of recombinant human lymphotoxin (rhLT) was studied in vitro. The simultaneous addition of 0.02 mM or 0.5 mM BSO and rhLT slightly augmented the inhibition of Meth A tumor cell proliferation. Similar tendency was observed when the target cells were treated with 0.02 mM or 0.5 mM BSO for 24 hr prior to the addition of rhLT. A marked augmentation of the antiproliferative effect was obtained when the target cells were treated in vitro with 0.005 mM or 0.02 mM BSO prior to the addition of 0.02 mM or 0.1 mM BSO and rhLT. The addition of ACNU simultaneously with rhLT to BSO-treated cells also augmented the antiproliferative effect. These results suggest that the glutathione redox cycle is closely related to the mechanism of LT-induced cytotoxicity.     

Effect of tetracaine and lidocaine on insulin release in isolated mouse pancreatic islets

The effect of tetracaine and lidocaine on insulin secretion and glucose oxidation by islets of ob/ob-mice was measured. Tetracaine, at a concentration of 1 microM to 0.1 mM, did not markedly influence the basal (3 mM glucose) insulin secretion, whereas 0.5-3.5 mM induced a marked increase. At 7 mM glucose, there was a dose-dependent increase with 0.1-2.5 mM tetracaine. Insulin release induced by 20 mM glucose was potentiated by 0.1 mM and 0.5 mM tetracaine, but this effect disappeared at 1 mM tetracaine. The stimulatory effect of 0.5-1 mM tetracaine on basal insulin release was blocked by the secretory inhibitors, adrenaline (1 microM), clonidine (1 microM) and by Ca2+-deficiency, but the stimulation by 3.5 mM tetracaine was not reduced by 1 microM clonidine or Ca2+ deficiency. Atropine (10 microM) did not affect the stimulation by 0.5 mM tetracaine at 3 mM glucose or by 0.25 mM tetracaine at 20 mM glucose. Tetracaine, at 0.1 mM, potentiated the secretory stimulation of 20 mM L-leucine, 20 mM D-mannose, or 1 microM glibenclamide. Mannoheptulose, 10 mM, abolished the combined effects of 0.1 mM tetracaine and 10 mM glucose. Lidocaine, 1-5 mM, stimulated basal insulin release, but 1 microM-1 mM of the drug did not affect glucose-induced (20 mM glucose) insulin release and 5 mM lidocaine inhibited glucose stimulation. The oxidation of 10 mM D-[U-14C]glucose was slightly enhanced by 0.1 and 1 mM tetracaine. The results indicate that tetracaine and lidocaine, at certain concentrations, can induce insulin release and that tetracaine potentiates secretion induced by other secretagogues. It is concluded that these effects may be associated with beta-cell functions related to the adrenergic receptors but probably not to cholinergic receptors.     

Various kinetic properties of beta-glucosidase cloned from Clostridium thermocellum in E. coli

The kinetics of pNPG, pNPX and cellobiose hydrolysis by beta-glucosidase cloned from C. thermocellum into E. coli was studied. The V values for these substrate hydrolysis are 102, 357 and 6.7 mumols/min/mg protein, respectively; Km are 0.44 mM, 50 mM and 100 mM, respectively, sigma-Gluconolactone inhibits the hydrolysis of all substrates according to a competitive mechanism with Ki of 0.032 mM, 6.0 mM and 0.25 mM, respectively. Glucose inhibits the hydrolysis of pNPG and pNPX also via a competitive mechanism with Ki of 10 mM and 37 mM, while cellobiose--via a mixed type mechanism with Ki of 110 mM and 350 mM. The existence of separate adsorption sites for each substrate and of a common catalytic site for pNPG and pNPX hydrolysis is supposed.     

Ileal resection and dietary content of sucrose influence the intestinal uptake of monosaccharides

The intestinal uptake of 0.5 and 40 mM glucose, galactose, and 3-O-methyl glucose (3-O-MG) was examined in vitro in rabbits fed a high (HS) or a low (LS) sucrose diet. In animals with an intact intestinal tract, the jejunal uptake of 0.5 mM 3-O-MG was unaffected by the dietary content of sucrose, whereas the uptake of 40 mM 3-O-MG was lower in LS than HS. The uptake of 40 mM galactose was higher in LS than HS and the uptake of 0.5 mM galactose was similar in HS and LS, whereas the uptake of 0.5 mM but not 40 mM glucose was lower in LS than HS. In animals subjected 6 weeks previously to an ileal resection, the adaptive changes in the jejunal uptake of the hexoses in response to alterations in the dietary content of sucrose differed from the changes observed in rabbits with an intact intestinal tract. For example, feeding HS to ileal resected animals was associated with increased jejunal uptake of 40 mM galactose, decreased uptake of 40 mM glucose, and unchanged uptake of 40 mM 3-O-MG; whereas in control animals with an intact intestinal tract, feeding HS resulted in increased uptake of 40 mM 3-O-MG, decreased uptake of 40 mM galactose, and no change in the uptake of 40 mM glucose. A similar adaptive pattern was noted in the jejunum and ileum for the effect of dietary sucrose on the uptake of 0.5 and 40 mM glucose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium enhances the hemolytic action of bile salts

The lysis of human erythrocytes by bile salts in buffer containing isotonic saline was dramatically enhanced by the addition of 5-10 mM calcium chloride. All bile acids tested showed this effect, with a marked increase in lysis occurring at 0.75 mM for deoxycholate, 1 mM for chenodeoxycholate, 2.5 mM for ursodeoxycholate and 5.5 mM with cholate in the presence of 10 mM calcium chloride. The effect appeared to be specific for calcium; strontium chloride and magnesium chloride gave no stimulatory effect. The increased lysis of the erythrocytes in the presence of 1 mM deoxycholate and 1-10 mM calcium chloride was not associated with increased uptake of the bile salt by the cells (measured with [14C]deoxycholate). Using erythrocytes previously labelled with [3H]cholesterol, there was no evidence of an enhanced removal of that membrane component in the presence of calcium and deoxycholate, compared to deoxycholate alone. The sensitivity of the cells to the effect of calcium in the presence of 1 mM deoxycholate increased with the length of time of their storage at 4 degrees C. The sensitivity returned to that of fresh cells after incubation at 37 degrees C with 30 mM adenosine plus 25 mM glucose, but this treatment did not further diminish the lysis. Lysis in the presence of 10 mM calcium chloride and 1 mM deoxycholate was partially blocked by increasing the KCl concentration at the expense of NaCl. The maximum effect occurred with a buffer comprising 100 mM KCl/50 mM NaCl. A more dramatic reduction in the lysis followed the incorporation of the calcium chelator, quin2, into the cells. The lysis induced by 1 mM deoxycholate in the presence of calcium was reduced by 80% in quin-2-loaded cells compared to controls. The data suggest that bile acids can promote the influx of calcium into erythrocytes, leading to lysis as a result of the efflux of intracellular potassium and/or the uptake of sodium from the incubation medium. The data further suggest that cellular effects may occur at lower bile acid concentrations than that thought to be required for detergent damage.     

Effect of Fructose Supplementation on Sorbitol Accumulation and myolnositol Metabolism in Cultured Neuroblastoma Cells Exposed to Increased Glucose Concentrations

Aldose reductase activity is increased in neuroblastoma cells grown in media containing 30 mM fructose and/or 30 mM glucose. Neuroblastoma cells cultured in media supplemented with increased concentrations of glucose and fructose amass greater amounts of sorbitol than do cells exposed to media containing only high glucose concentrations. The increase in sorbitol content is dependent on the fructose and glucose concentration in the media. The increase in sorbitol content caused by exposing neuroblastoma cells to media containing 30 mM glucose/30 mM fructose is due to a protein synthesis sensitive mechanism and not to an alteration in the redox state. The addition of sorbinil to media containing 30 mM glucose blocks the increase in sorbitol content. In contrast, sorbinil treatment of media containing 30 mM glucose/30 mM fructose does not totally block the increase in sorbitol levels. myo-Inositol accumulation and incorporation into inositol phospholipids and intracellular myo-inositol content are decreased in cells chronically exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose compared to cells cultured in unsupplemented media or media containing 30 mM fructose. However, maximal depletion of myo-inositol accumulation and intracellular content occurs earlier in cells exposed to media containing 30 mM glucose/30 mM fructose than in cells exposed to media supplemented with 30 mM glucose. Sorbinil treatment of media containing 30 mM glucose/30 mM fructose maintains cellular myo-inositol accumulation and incorporation into phospholipids at near normal levels. myo-Inositol content in neuroblastoma cells chronically exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose recovers within 72 h when the cells are transferred to unsupplemented media or media containing 30 mM fructose. In contrast, the sorbitol content of cells previously exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose then transferred into media containing 30 mM fructose remains elevated compared to the sorbitol content of cells transferred into unsupplemented media. These data suggest that fructose may be activating or increasing sorbinil-resistant aldose reductase activity as well as partially blocking sorbitol dehydrogenase activity. The presence of increased concentrations of fructose in combination with increased glucose levels may enhance alterations in cell metabolism and properties due to increased sorbitol levels.     

Production of weak acid by anaerobic fermentation of soil and antifungal effect

Acetic acid and butyric acid were produced by the anaerobic fermentation of soil mixed with wheat or rice bran. The concentration of acetic acid produced in the wheat and rice bran-treated soil was 31.2 mM and 8 mM, respectively, whereas the concentration of butyric acid in the wheat and rice bran-treated soil was 25.0 mM and 8 mM, respectively. The minimal fungicidal concentration (MFC) for all the fungal strains was 40-60 mM acetic acid, 20-40 mM butyric acid, and 40-60 mM mixture of acetic acid: butyric acid (1:1, v/v). Consequently, the efficacy of mixing wheat-bran with soil to control soil diseases was demonstrated.     

The mechanism of the effect of K+ on the steroidogenesis of rat zona glomerulosa cells of the adrenal cortex: role of cyclic AMP

The effects of various concentrations of extracellular K+ (3.6-13 mM) on the steroid (corticosterone and aldosterone) and cyclic AMP outputs of capsular cells (95% zona glomerulosa) of the rat adrenal cortex were studied at different concentrations of extracellular Ca2+. Small amounts of EGTA (50 microM) were added to reduce the free Ca2+ concentrations effectively to zero at the lowest possible total Ca2+ concentration. At a total extracellular concentration of 2.5 mM Ca2+, in 27 experiments the mean values of the steroid and cAMP outputs showed a maximum at 8.4 mM K+. The increase in steroid and cAMP outputs at 5.9, 8.4 and 13 mM K+ compared with that at 3.6 mM were highly significant (p less than 0.01). The overall correlation of either corticosterone or aldosterone with cAMP outputs was also highly significant and was even better from 3.6 to 8.4 mM K+. Lowering the effective free concentration of Ca2+ to zero decreased the steroid and cAMP outputs significantly at all K+ concentrations, and no output was then significantly higher than at 3.6 mM. With the pooled data on outputs at all total Ca2+ (2.5, 0.5, 0.25, 0.10, 0.05 and 0.0 mM) and K+ (3.6, 5.9, 8.4 and 13 mM) concentrations, the correlation of either steroid with cAMP outputs was highly significant (but again optimally from 3.6 to 8.4 mM K+). Nifedipine (10(-6) to 10(-4) M) was added to the incubations with the aim of specifically inhibiting Ca2+ influx at total extracellular Ca2+ concentrations of 2.5, 1.25 and 0.25 mM and with the usual K+ concentrations. The cAMP outputs were reduced at all K+ concentrations above 3.6 mM K+. The effect was highly significant at 10(-4) M nifedipine and a total Ca2+ of 1.25 mM, which with the incubation conditions used, corresponds to the free Ca2+ concentrations in vivo. These results indicate that cAMP plays a significant role in the stimulation of steroid output by K+ particularly between 3.6 and 8.4 mM K+. In this range of K+ concentrations the stimulation of cAMP seems to be controlled by increases in Ca2+ influx. The correlation of steroid and cAMP output at the higher K+ concentrations (between 8.4 and 13 mM K) and at the various total Ca2+ concentrations is less significant. Also, with all concentrations of added nifedipine there is an 'anomalous' increase in steroid output at 13 mM K+ and at total Ca2+ concentrations of 2.5 and 1.25 mM. However, at the same K+ concentrations and at 0.25 mM Ca2+, nifedipine decreases steroid outputs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intravital survival and deformability of weakly amidinated rat erythrocytes]

Rat erythrocytes previously labelled with 51Cr were treated with 0.1 mM and 0.5 mM dimethyladipic imidate (ADE) pH 9.5, respectively, or with borate buffer pH 9.5 for 15 minutes each. After reinjection into rats of amidinated or nonamidinated erythrocytes their elimination form the streaming blood was followed for 50 days (0.1 mM ADE) or 38 days (0.5 mM ADE). During the 1st day about 10% of amidinated erythrocytes were sequestered. There was no increased initial elimination rate of borate incubated red blood cells detectable. During the following period the elimination rate of erythrocytes amidinated with 0.5 mM ADE was slightly greater than in the case of erythrocytes treated with 0.1 mM ADE. The mean life span of 0.5 mM ADE-erythrocytes was shorter compared with the controls and with 0.1 mM ADE-erythrocytes. Red blood cells previously treated with 0.5 mM ADE are significantly less deformable. Presumably, the stiffness of amidinated erythrocytes causes their increased elimination rate.     

In vitro binding of 70S ribosomes to membrane structures of Escherichia coli

It was found that the maximal disattachment of the ribosomes from the membrane structures is observed upon their treatment with 10 mM tris-HCl buffer, pH 7.5, containing 250 mM sucrose, 750 mM KCl, 5 mM magnesium acetate and 1 mM EDTA or puromycin. The most effective attachment of ribosomes to the membrane occurs in 10 mM tris-HCl buffer, pH 7.5, containing 5% sucrose and Mg2+. The increase of Mg2+ concentration in the medium from 0.5 mM up to 1 mM results in a 2-fold increase of the ribosomes bound to the membranes. The concentration of the ribosomal material involved in the reaction is very essential for ribosome binding to the membranes. The amount of ribosomes bound to the membranes increases proportionally to the increase of the ribosome concentration in the reaction mixture.     

Regeneration of bacteriorhodopsin in mixed micelles

Regeneration of bacteriorhodopsin from bacterioopsin and all-trans-retinal was studied in a mixed micelle system consisting of dodecyl sulfate, CHAPS and a water-soluble phospholipid dihexanoylphosphatidylcholine (hex2-PhosChol). Regeneration to approximately 40,000 M-1.cm-1 extinction at 550 nm (epsilon 550) was obtained with either 2.3 mM or 6.5 mM CHAPS along with 6.9 mM dodecyl sulfate and 4.5 mM hex2-PhosChol in 0.16 M NaCl and 40 mM phosphate (pH 6.0). Without CHAPS, the regeneration in 4.5 mM Hex2-PhosChol gave epsilon 555 = 27,800; without PhosChol, the 1:3 CHAPS/dodecyl sulfate mixture gave epsilon 550 approximately 20,000; and without PhosChol the nearly equimolar CHAPS/dodecyl sulfate mixture gave epsilon 550 approximately 10,000. The composition of the mixed micelles was estimated from fluorescence spectroscopy using pyrene butyryl hydrazine. The molecular weight was estimated by molecular seive chromatography to be 87,100 for 2.3 mM CHAPS, 6.9 mM dodecyl sulfate and 0.67 mM hex2-PhosChol; and 83,200 for 7.0 mM CHAPS, 6.9 mM dodecyl sulfate, and 1.1 mM hex2-PhosChol. These results are consistent with the idea that at low concentrations of CHAPS and dodecyl sulfate, CHAPS organizes the dodecyl sulfate into disk shaped bilayer micelles that are favorable for bacterioopsin refolding. However, a high concentration of either detergent inhibits regeneration. Added hex2-PhosChol can overcome the inhibitory effects of high concentrations of either CHAPS or dodecyl sulfate.     

Conversion of glycerate to serine in intact spinach leaf peroxisomes

Intact spinach (Spinacia oleracea L.) leaf peroxisomes converted glycerate to serine in the presence of NAD and alanine. The reaction proceeded optimally at pH9. Addition of oxaloacetate or alpha-ketoglutarate plus aspartate enhanced the conversion about three-fold. Alteration of the concentration of one of the reaction components, consisting of 2 mM glycerate, 0.2 mM NAD, 0.5 mM oxaloacetate, and 2 mM alanine, revealed half-saturation constants of 0.45 mM for glycerate, 0.06 mM for NAD, 0.02 mM for oxaloacetate, and 0.33 mM for alanine. The conversion proceeded with the formation of hydroxypyruvate followed by serine; hydroxypyruvate did not accumulate to a high amount in the presence or absence of alanine. The amino group donor could be alanine (half-saturation constant, 0.33 mM), glycine (0.45 mM), or asparagine (0.67 mM); the three amino acids produced roughly similar Vmax values. The results indicate that, in the conversion of glycerate to serine, the transamination is catalyzed by a hydroxypyruvate aminotransferase with characteristics unknown among all other studied leaf peroxisomal aminotransferases. The peroxisomal membrane is sparsely permeable to NAD/NADH, and the participation of the peroxisomal malate dehydrogenase in an electron shuttle system across the membrane in the regeneration of NAD/NADH is suggested.     

Substrate specificity and kinetic mechanism of Escherichia coli ribulokinase.

L-ribulokinase is unusual among kinases since it phosphorylates all four 2-ketopentoses with almost the same k(cat) values. The K(m)'s differ, however, being 0.14 mM for L- and 0.39 mM for d-ribulose and 3.4 mM for l- and 16 mM for d-xylulose. In addition, L-arabitol is phosphorylated at C-5 (K(m) 4 mM) and ribitol (adonitol) is phosphorylated to D-ribitol-5-phosphate (K(m) 5.5 mM), but D-arabitol, xylitol, and aldopentoses are not substrates. The K(m)'s for MgATP depend on the substrates, being 0.02 mM with L-ribulose, 0.027 mM with D-ribulose and L-xylulose, and 0.3-0.5 mM with the other substrates. In the absence of a sugar substrate there is an ATPase with K(m) of 7 mM and k(cat) 1% of that with sugar substrates. The initial velocity pattern is intersecting, and MgAMPPNP is competitive vs MgATP and uncompetitive vs L-ribulose. L-Erythrulose is competitive vs L-ribulose and when MgATP concentration is varied induces substrate inhibition which is partial. These data show that the mechanism is random, but there is a high level of synergism in the binding of sugar and MgATP, and the path in which the sugar adds first is strongly preferred.     

Ionic determinants of spontaneous activity in clusters of cultured cardiac cells from newborn rats.

The spontaneous activity of cell clusters derived from ventricle cells of newborn rats was studied using a recording television microscope. The influence of varying concentrations of sodium, potassium, calcium, tetrodotoxin (TTX), and that of 2 mM MnCl2 was tested. The spontaneous activity of the cell clusters persisted in TTX but it was abolished by Mn. The beating rate increased when [Ca]0 and [Na]0 were changed from 0.3 mM to 3.0 mM and from 30 mM to 75 mM; it decreased with a change of [Na]0 from 75 mM to 142 mM. It is concluded that electrogenesis in their behavior to very young embryonic rat heart cells or cells of the rabbit sinoauricular node.     

The effect of storage on the kinetic properties of sheep hepatic pyruvate kinase

The kinetic properties of purified sheep hepatic pyruvate kinase change upon storage. Assayed at 0.5 mM fructose-1,6-diphosphate and 2 mM ADP, saturation of fresh enzyme with phosphoenolpyruvate is hyperbolic, with KPEP = 0.1 mM (pH 7.5, and 30 degrees C). Under similar conditions enzyme stored at -20 degrees C for 1 week or more yields a nonlinear Lineweaver-Burk plot for PEP. The data may be accounted for by the appearance of two enzymic forms with identical turnover numbers, but different KPEP (0.035 +/- 0.005 and 12.4 +/- 0.6 mM). Storage also increases the concentration of fructose-1,6-diphosphate required for maximal activation from nanomolar to millimolar levels. Assayed at 2 mM ADP and 2 mM PEP, the apparent KFDP is 10 mM. Preincubation of stored enzyme with PEP in the presence of mercaptoethanol leads to significant reversion to original kinetic properties. Available data suggest that the storage-dependent change in kinetic behavior rises from changes in subunit conformation and not from dissociation into subunits.     

Preparation and activity of guanidinated or acetylated erabutoxins.

1. The effects of phenylalanine and its metabolites (phenylacetate, phenethylamine, phenyl-lactate, o-hydroxyphenylacetate and phenylpyruvate) on the activity of 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) 3-oxo acid CoA-transferase (EC 2.8.3.5) and acetoacetyl-CoA thiolase (EC 2.3.1.9) in brain of suckling rats were investigated. 2. The 3-hydroxybutyrate dehydrogenase from the brain of suckling rats had a Km for 3-hydroxybutyrate of 1.2 mM. Phenylpyruvate, phenylacetate and o-hydroxyphenylacetate inhibited the enzyme activity with Ki values of 0.5, 1.3 and 4.7 mM respectively. 3. The suckling-rat brain 3-oxo acid CoA-transferase activity had a Km for acetoacetate of 0.665 mM and for succinyl (3-carboxypropionyl)-CoA of 0.038 mM. The enzyme was inhibited with respect to acetoacetate by phenylpyruvate (Ki equals 1.3 mM) and o-hydroxyphenylacetate (Ki equals 4.5 mM). The reaction in the direction of acetoacetate was also inhibited by phenylpyruvate (Ki equals 1.6 mM) and o-hydroxyphenylacetate (Ki equals 4.5 mM). 4. Phenylpyruvate inhibited with respect to acetoacetyl-CoA both the mitochondrial (Ki equals 3.2 mM) and cytoplasmic (Ki equals 5.2 mM) acetoacetyl-CoA thiolase activities. 5. The results suggest that inhibition of 3-hydroxybutyrate dehydrogenase and 3-oxo acid CoA-transferase activities may impair ketone-body utilization and hence lipid synthesis in the developing brain. This suggestion is discussed with reference to the pathogenesis of mental retardation in phenylketonuria.     

Inhibition of H+ Extrusion by Phosphocreatine in Candida albicans

Vanadate, a potent inhibitor of P-type ATPases, reduces the electrochemical gradient considerably. H⁺-extrusion in cells of Candida albicans, a pathogenic yeast, was strongly inhibited in the presence of 25mM phosphocreatine (PCr) by about 83%. H⁺-extrusion was further inhibited by 25 mM PCr in the presence of vanadate; 89% with 1 mM, 92% with 2 mM and 99% with 5 mM vanadate. 2 mM vanadate caused 90%, 92% and 96% inhibition in the presence of 20 mM, 30 mM and 40 mM PCr, respectively. Creatine (Cr) had a negligible effect on H⁺ - extrusion. The inhibition caused by 1 mM, 2 mM and 5 mM vanadate alone was 66%, 77% and 88%, respectively. PCr and vanadate inhibit proton extrusion with almost equal magnitude. It can be concluded that phosphate moiety of PCr interacts with the ATPase and is similar to vanadate interaction. Since PCr is having such a drastic inhibitory effect on ATPase activity we can say that it is playing a significant role in holding a check on this pathogenic fungus in healthy human hosts.