Inhibition of rat liver and kidney arginase by cadmium ion

Cadmium ion activates arginase from many species of organisms but is an inhibitor of arginase from many other species. The purpose of this study was to investigate the inhibition of rat liver and kidney arginase by cadmium ion. Rat kidney arginase was inhibited by much lower concentrations of cadmium ion than rat liver arginase. Cadmium ion was a mixed noncompetitive inhibitor of both rat liver and kidney arginase. Cadmium ion enhanced the substrate activation of rat kidney arginase while still inhibiting the enzyme. Cadmium ion prevented the substrate inhibition of rat kidney arginase by fluoride while still inhibiting the enzyme. Cadmium ion also inhibited rat kidney arginase in the presence of manganese ion.     

Inhibition of rat liver and kidney arginase by cadmium ion

Cadmium ion activates arginase from many species of organisms but is an inhibitor of arginase from many other species. The purpose of this study was to investigate the inhibition of rat liver and kidney arginase by cadmium ion. Rat kidney arginase was inhibited by much lower concentrations of cadmium ion than rat liver arginase. Cadmium ion was a mixed noncompetitive inhibitor of both rat liver and kidney arginase. Cadmium ion enhanced the substrate activation of rat kidney arginase while still inhibiting the enzyme. Cadmium ion prevented the substrate inhibition of rat kidney arginase by fluoride while still inhibiting the enzyme. Cadmium ion also inhibited rat kidney arginase in the presence of manganese ion.     

In vitro inhibition of alkaline phosphatase activities from intestine, bone, liver, and kidney by phenobarbital.

A kinetic study of the inhibition of several alkaline phosphatase (AP isoenzyme activities by phenobarbital was carried out using p-nitrophenylphosphate (10 mM) as a substrate at pH 9.8 in a 300-mM Hepes buffer. AP from bovine kidney, calf intestine, bovine liver, and rat bone was used. Over a phenobarbital concentration range of 20-400 mM, all these isoenzymes were inhibited in an uncompetitive manner with a Ki of 200 mM for intestinal AP, and in a linear mixed-type manner for all the other isoenzymes tested. The Ki values were 10, 40 and 55 mM for kidney, bone and liver AP, respectively. The use of 15 mM carbonate-bicarbonate or 400 mM diethanolamine buffer did not modify the degree of inhibition of intestinal AP activity. Dixon plots of the reciprocal of reaction velocity versus inhibitor concentration either at different substrate concentration or at different DEA concentration indicate uncompetitive inhibition for the intestinal enzyme. This in vitro inhibitory effect of phenobarbital is in contrast to its in vivo stimulating action on AP. However, in the whole animal, the effects of phenobarbital administration probably represent the sum of multiple effects.     

Fluoride effects on the plasma membrane ATPase of sugarbeet

Fluoride, a common air pollutant long known as a toxicant to many plant processes, inhibits mitochondrial, chloroplast and tonoplast ATPases. In the present study, the effects of fluoride at various substrate concentrations on the plasma membrane ATPase of sugarbeets (Beta vulgaris L.) were investigated. The plasma membrane ATPase was inhibited by lower concentrations (5 mM) of fluoride than the above indicated ATPases. The amount of inhibition due to fluoride increased with increasing concentrations of free Mg²⁺ in the reaction medium. The data suggest that fluoride inhibition of the plasma membrane ATPase is at the active site of the enzyme and occurs via a magnesium-fluoro-complex.     

Purification, properties and alternate substrate specificities of arginase from two different sources: Vigna catjang cotyledon and buffalo liver

Arginase was purified from Vigna catjang cotyledons and buffalo liver by chromatographic separations using Bio-Gel P-150, DEAE-cellulose and arginine AH Sepharose 4B affinity columns. The native molecular weight of an enzyme estimated on Bio-Gel P-300 column for Vigna catjang was 210 kDa and 120 kDa of buffalo liver, while SDS-PAGE showed a single band of molecular weight 52 kDa for cotyledon and 43 kDa for buffalo liver arginase. The kinetic properties determined for the purified cotyledon and liver arginase showed an optimum pH of 10.0 and pH 9.2 respectively. Optimal cofactor Mn++ ion concentration was found to be 0.6 mM for cotyledon and 2 mM for liver arginase. The Michaelis-Menten constant for cotyledon arginase and hepatic arginase were found to be 42 mM and 2 mM respectively. The activity of guanidino compounds as alternate substrates for Vigna catjang cotyledon and buffalo liver arginase is critically dependent on the length of the amino acid side chain and the number of carbon atoms. In addition to L-arginine cotyledon arginase showed substrate specificity towards agmatine and L-canavanine, whereas the liver arginase showed substrate specificity towards only L-canavanine.     

Inhibition of Water Melon (Citrullus vulgaris) Urease by Fluoride

Effect of fluoride on the activity of purified urease from seeds of watermelon (Citrullus vulgaris) was studied. Fluoride exhibited a concentration dependent inhibition both in presenceand absence ofthe substrate. The inhibition was non-competitive. Addition of 8mM β-mercaptoethanol gradually abolished the fluoride inhibition. β-mnercaptoethanol, in presence of fluoride, also exhibited a concentration dependent suppression of inhibition caused by fluoride. The significance of these observations is discussed.     

Rat Mammary Arginase: Isolation and Characterization

The extrahepatic arginase, AII, from rat mammary gland was isolated and its properties investigated and compared with those of the hepatic arginase, AI. Mammary arginase activity increased 300% at mid-lactation, an increase unaccompanied by an increase in liver arginase activity. Mammary gland contained two isozymes, separable by ion exchange chromatography. The major form, AII, was purified 103-fold and antisera were raised against it. A 1300-fold purification was achieved temporarily but the enzyme was unstable. Arginase AII was kinetically similar to AI: both had pH optima of 10 and K_ms for L-arginine of 12-14 mM. Arginase AII differed from AI in having a near-neutral pI and a slightly larger subunit size (39,800 Da compared to 38,900 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)). Solution immunoprecipitation studies revealed that virtually all of the arginase present in liver was type AI, whereas kidney and mammary gland contained both isozymes. Western immunoblotting showed that the amount of immunoreactive mammary arginase AII protein increased at mid-lactation in parallel with the increase in activity. This suggests that the elevated arginase activity is due to de novo protein synthesis and/or reduced protein degradation, rather than activation of arginase.     

Activation of rat adipocyte plasma membrane adenylate cyclase by sodium azide

The adenylate cyclase of rat adipocyte plasma membrane is stimulated by sodium azide with a half maximal activation of 100–150% occuring at 50 mM NaN₃. Studies of the effects of azide and fluoride indicate different mechanisms of stimulation of the enzyme by these ions. Comparable stimulation of the activity is obtained by 100 mM NaN₃ or 10 mM NaF but unlike azide, higher concentrations of fluoride cause inhibition of the enzyme. Fluoride activated adenylate cyclase is further stimulated by azide. Epinephrine stimulation of the enzyme is absent in the presence of fluoride but the hormone enhances the activity in the presence of azide. Reversal of the inhibitory action of GTP on adenylate cyclase by epinephrine is demonstrated even in the presence of azide but not in the presence of fluoride.     

Quasi-irreversible inhibition of enolase of Streptococcus mutans by flouride

Abstract Fluoride at concentrations greater than 0.01 mM was found to be a quasi-irreversible inhibitor of enolase of permeabilized cells of Streptococcus mutans GS-5 and also of isolated yeast enolase. The inhibition appeared to be of the type that has been described for P-ATPases, but was not dependent on added Al³⁺ or Be²⁺ ions. Fluoride inhibition of enolase was not reversed by repeatedly washing the permeabilized cells in chilled fluoride-free medium but could be reversed by the product, phosphoenolpyruvate, or by very high levels of the substrate, 2-phosphoglycerate. Irreversible inhibition of glycolysis was not evident after fluoride treatment of intact cells, washing to remove unbound or loosely bound fluoride and addition of glucose, presumably because intracellular levels of phosphoenolpyruvate were sufficiently high to preclude irreversible fluoride inhibition of enolase.     

Inhibition of cholinesterases by fluoride in vitro

1. Series of colorimetric dynamic assays allowed the study of the inhibition of cholinesterases by F(-) ions in vitro, by using, as sources of enzyme, whole human blood, human serum, homogenized rat brain and two preparations of red blood cells (human and bovine) whose enzymic purity was ascertained. 2. The first evidence of inhibition of human serum pseudocholinesterase by fluoride was noticed at 15-25mum-fluoride. Ten times as much fluoride was needed to start inhibition of acetylcholinesterase of the red blood cells. 3. The action of fluoride on the enzymic reaction was immediate. The reversibility of the inhibition was shown by dialysis and dilution. 4. Kinetic measurements showed that the inhibition under study was not dependent on the substrate concentration and was of the uncompetitive type, similar to that observed in the presence of a heavy metal (cadmium). 5. The activity of serum cholinesterase did not change in the absence of Mg(2+) and Ca(2+) ions. Fluoride was shown to inhibit the enzyme in the absence of these ions as well as of phosphate. 6. Fluoride could inhibit cholinesterases in the presence of three different substrates and had no action on the non-enzymic hydrolysis. 7. It is thought that the halide is bound reversibly to the enzyme molecule, with the probable exclusion of the active site, but no firm conclusion could be reached on this point.     

Fluoride inhibition of Klebsiella aerogenes urease: mechanistic implications of a pseudo-uncompetitive, slow-binding inhibitor

Klebsiella aerogenes urease uses a dinuclear nickel active site to catalyze the hydrolysis of urea. Here, we describe the steady-state and pre-steady-state kinetics of urease inhibition by fluoride. Urease is slowly inhibited by fluoride in both the presence and absence of substrate. Steady-state rate studies yield parallel double-reciprocal plots; however, we show that fluoride interaction with urease is not compatible with classical uncompetitive inhibition. Rather, we propose that fluoride binds to an enzyme state (E) that is in equilibrium with resting enzyme (E) and produced during catalysis. Fluoride binding rates are directly proportional to inhibitor concentration. Substrate reduces both the rate of fluoride binding to urease and the rate of fluoride dissociation from the complex, consistent with urea binding to E and E.F in addition to E. Fluoride inhibition is pH-dependent due to a protonation event linked to fluoride dissociation. Fluoride binding is pH-independent, suggesting that fluoride anion, not HF, is the actual inhibitor. We assess the kinetic results in terms of the known protein crystal structure and evaluate possible molecular interpretations for the structure of the E state, the site of fluoride binding, and the factors associated with fluoride release. Finally, we note that the apparent uncompetitive inhibition by fluoride as reported for several other metalloenzymes may need to be reinterpreted in terms of fluoride interaction with the corresponding E states.     

In hepatocytes the regulation of NOS-2 activity at physiological l-arginine levels suggests a close link to the urea cycle

High-output synthesis of nitric oxide (NO) by the inducible isoform of NO-synthases (NOS-2) plays an important role in hepatic pathophysiological processes and may contribute to both organ protection and organ destruction during inflammatory reactions. As they compete for the same substrate, L-arginine, an interdependence of NOS-2 and arginase-1 has been repeatedly observed in cells where arginase-1 is cytokine-inducible. However, in hepatocytes, arginases are constitutively expressed and thus, their impact on hepatic NOS-2-derived NO synthesis as well as the influence of L-arginine influx via cationic amino acid transporters during inflammatory reactions are still under debate. Freshly isolated rat hepatocytes were cultured for 24h in the presence of various L-arginine concentrations with or without cytokine addition and nitrite and urea accumulation in culture supernatants was measured. We find that both, cytokine-induced NOS-2 and arginase activities strongly depend on extracellular L-arginine concentrations. When we competed for L-arginine influx via the cationic amino acid transporters by addition of L-lysine, we find a 60-70% inhibition of arginase activity without significant loss of NOS-2 activity. Addition of L-valine, as an arginase inhibitor, leads to a 25% increase in NO formation and an 80-90% decrease in arginase activity. Interestingly, product inhibition of arginase and competitive inhibition of CATs through the addition of L-ornithine leads to a highly significant increase in hepatocytic NOS-2 activity with a concomitant and complete abolishment of its dependence on extracellular L-arginine concentrations. In conclusion, hepatocytic NOS-2 activity shows a surprising pattern of dependence on exogenous L-arginine concentrations. Inhibition and competition experiments suggest a relatively tight link of NOS-2 and urea cycle activities. These data stress the hypothesis of a metabolon-like organization of the urea cycle together with NOS-2 in hepatocytes as excess L-ornithine will be metabolized to l-arginine and thereby increases NO production.     

Regulation of macrophage physiology by L-arginine: role of the oxidative L-arginine deiminase pathway

The L-arginine content of the extracellular fluid in sites of predominant macrophage infiltration is reduced below plasma levels due to the activity of macrophage-derived arginase. Investigation of the effects of altered L-arginine availability on macrophage physiology reveals that culture of rat peritoneal macrophages in media containing L-arginine in the concentrations present in inflammatory lesions (less than 0.1 mM) enhances activation-associated functions. In contrast, culture in the higher L-arginine concentrations found in standard tissue culture media (0.4 to 1.2 mM) suppresses most macrophage functions (superoxide production, phagocytosis, and protein synthesis). An exception is the tumor cytotoxicity of Corynebacterium parvum-elicited macrophages which is enhanced by culture in supraphysiologic concentrations of L-arginine. Work reported here investigated the mechanisms for these L-arginine-dependent effects and, more specifically, the role of the recently described oxidative L-arginine deiminase pathway in the regulation of macrophage physiology. Overnight culture of resident or C. parvum-elicited peritoneal macrophages in media containing increasing concentrations of L-arginine (6 microM to 1 mM) resulted in: inhibition of electron transport chain activity (resident and C. parvum-elicited macrophages), increased lactate production (resident macrophages), and decreased ATP content (resident and C. parvum-elicited macrophages). In line with these findings, viability was markedly decreased after 2 days of culture when the initial L-arginine concentration was greater than or equal to 0.1 mM. As shown before, increasing media concentrations of L-arginine were associated with suppression of superoxide production and cytotoxicity in resident macrophages, and with reduced superoxide production and increased cytotoxicity in C. parvum-elicited macrophages. All L-arginine-dependent metabolic and functional alterations, as well as the loss of viability, were prevented by NG-monomethyl-L-arginine, a specific inhibitor of the oxidative L-arginine deiminase pathway. These results demonstrate that flux of L-arginine through the oxidative L-arginine deiminase pathway results in the inhibition of oxidative metabolism in rat macrophages. This metabolic inhibition may, through alterations in the macrophage high energy phosphate stores, mediate the suppression of cell functions and result ultimately in cell death.     

Allosteric inhibition of rat liver and kidney arginase by copper and mercury ions

Two isozyme forms of arginase are found in the rat. All arginases are metalloenzymes which require manganese for activity. Many arginases are activated by cobalt and nickel ions and inhibited by heavy metal ions. The purpose of this study was to compare the effect of other heavy metal ions on the rat liver isozyme (arginase I) and the rat kidney isozyme (arginase II). The activation and inhibition of arginase I and II by metal ions were different. However, both isozymes were strongly inhibited by cupric and mercuric ions. The inhibition of arginase I by cupric and mercuric ions was increased greatly by preincubation of the enzyme with the metal ions. However, preincubation of arginase II by cupric and mercuric ions had little effect on the inhibition of the enzyme. Under certain conditions the kinetics of the inhibition of both arginases I and II by cupric and mercuric ions was nonlinear allosteric.     

Ketone body usage by mammals. Acetoacetate substrate inhibition of CoA transferase from various rat tissues

Succinyl-CoA: acetoacetate CoA transferases from rat kidney, heart, brain, and skeletal muscle display substrate inhibition by acetoacetate that is characterized by an “inversion concentration” of 4–6 mM acetoacetate, i.e., at acetoacetate concentrations greater than 5 mM inhibition is detectable. A similar effect is manifested with intact, uncoupled kidney mitochondria, suggesting that mitochondrial oxidation of ketone bodies can reflect CoA transferase kinetic properties with regard to acetoacetate inhibition. Since acetoacetate substrate inhibition of rat CoA transferase becomes apparent at concentrations that correspond to the plasma concentrations of total ketone bodies found during pathological ketosis, this substrate inhibotory effect may play a role in establishing the disturbed metabolic pattern of ketone bodies in diabetic animals.     

Arginase from rat fibrosarcoma. Purification and properties

Arginase from rat fibrosarcoma was purified about 1900-fold and its properties were compared with those of the enzyme from liver and kidney. Arginase from fibrosarcoma was a neutral protein of molecular weight 120,000 with a K_m value of 11 mM for arginine. The activation energy was 7.2 kcal/mol and the pH optimum was 10. The fibrosarcoma enzyme was immunologically different from that of the liver. The arginase from fibrosarcoma closely resembled the arginase from the kidney in its electrophoretic, kinetic and immunological properties.     

Studies on the kynurenine adminotransferase activity in rat liver and kidney.

The kynurenine aminotransferase activity of supernatant and mitochondrial fractions obtained from rat liver and kidney was studied with L-kynurenine and L-3-hydroxykynurenine as substrates. A substrate inhibition with L-kynurenine at concentrations higher than 6-7mM was observed with all four enzyme preparations. This did not happen with L-3-hydroxykynurenine as a substrate. Moreover, the liver mitochondrial enzyme shows a Km for pyridoxal phosphate 2-4 times smaller than the other preparations when assayed with L-3-hydroxykynurenine as a substrate. Therefore, the accumulation of xanthurenic acid and not of kynurenic acid in B6 deficiency could be related both to this high activity of liver mitochondrial kynurenine aminotransferase with L-3-hydroxykynurenine, even at small concentrations of B6, and to substrate inhibition observed with L-kynurenine and not with L-3-hydroxykynurenine.     

Assay and kinetics of arginase

A sensitive colorimetric assay for arginase was developed. Urea produced by arginase was hydrolyzed to ammonia by urease, the ammonia was converted to indophenol, and the absorbance was measured at 570 nm. The assay is useful with low concentrations of arginase (0.5 munit or less than 1 ng rat liver arginase) and with a wide range of arginine concentrations (50 microM to 12.5 mM). Michaelis-Menten kinetics and a Km for arginine of 1.7 mM were obtained for Mn2+-activated rat liver arginase; the unactivated enzyme did not display linear behavior on double-reciprocal plots. The kinetic data for unactivated arginase indicated either negative cooperativity or two types of active sites on the arginase tetramer with different affinities for arginine. The new assay is particularly well suited for kinetic studies of activated and unactivated arginase.     

The kinetics of inhibition of Vigna catjang cotyledon and buffalo liver arginase by L-proline and branched-chain amino acids

The effect of proline, isoleucine, leucine, valine, lysine and ornithine under standard physiological conditions, on purified Vigna catjang cotyledon and buffalo liver arginases was studied. The results showed that V. catjang cotyledon arginase is inhibited by proline at a lower concentration than buffalo liver arginase and the inhibition was found to be linear competitive for both enzymes. Buffalo liver arginase was more sensitive to inhibition by branched-chain amino acids than V. catjang cotyledon. Leucine, lysine, ornithine and valine are competitive inhibitors while isoleucine is a mixed type of inhibitor of liver arginase. We have also studied the effect of manganese concentration which acts as a cofactor and leads to activation of arginase. The optimum Mn2+ concentration for Vigna catjang cotyledon arginase is 0.6 mM and liver arginase is 2.0 mM. The preincubation period required for liver arginase is 20 min at 55 degrees C, the preincubation period and temperature required for activation of cotyledon arginase was found to be 8 min at 35 degrees C. The function of cotyledon arginase in polyamine biosynthesis and a possible role of branched chain amino acids in hydrolysis of arginine in liver are discussed.