Influence of ring- and side-chain substituents on the transamination of aromatic amino acids by two multispecific aspartate-aromatic aminotransferase isozymes purified from bushbean shoots

The breadth of substrate specificity shown by the multispecific aspartate-aromatic aminotransferase of bushbean (Phaseolus vulgaris) has been investigated by testing the ability of two cytosolic isozymes (I and II), purified from shoot tissue, to catalyse transamination reactions between a range of ring- and sidechain-substituted aromatic amino acids and 2-oxoglutarate. Ring-substituted phenylalanines were the most reactive substrates whereas ring-substitution in tyrosine or tryptophan resulted in transamination rates lower than those observed with the parent amino acids. All side chain-substituted analogues were found to be totally inactive. The highest activity shown by any ring-substituted phenylalanine was observed with the 4-amino- compound, followed closely by the 4-hydroxy- and 4-halogen-compounds. In contrast, 4-nitrophenylalanine was completely inactive. These trends were consistent for both isozymes I and II, but only isozyme II showed greatly enhanced activity over that found with the parent amino acid when certain ring-substituted analogues were tested. The varying capacity of the bushbean isozymes to utilize the present range of substituted amino acids is compared with previous reports on the substrate specificity shown by aspartate and aromatic aminotransferases isolated from mammalian and microbial systems. A model for the mechanism of activation observed with bushbean isozyme II in the presence of certain 4-substituted aromatic amino acids is proposed, based on current understanding of the nature of the active site of animal aspartate aminotransferases.     

Pyruvate kinase isozymes in adult tissues and eggs of Rana pipiens. Comparative studies on the properties of the different isozymes

The properties of the isozymes of pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) found in unfertilized frog egg have been compared to those found in adult tissues of Rana pipiens. Chromatographic, kinetic, and electrophoretic data indicate that, of the five electrophoretic forms found in egg, the isozyme with the least anodic mobility (isozyme I) is the same molecular species as the only isozyme found in heart, and the egg isozyme with the greatest anodic mobility (isozyme V) is identical to the major isozyme found in liver.The activity of egg isozyme I was markedly inhibited by the antibody to the skeletal muscle enzyme, which has been shown previously to cross-react with the cardiac enzyme, but was unaffected by the antibody to liver isozyme V; the opposite effects were observed with egg isozyme V. The antibody to the skeletal muscle enzyme inhibited egg isozymes II > III > IV whereas the antibody to the liver enzyme gave the reverse inhibitory pattern, e.g., isozyme IV > III > II.In vitro dissociation-reassociation of mixtures of isozyme I and V led to the formation of the other three isozymes. Similar experiments performed individually with either egg isozyme III or IV resulted in the production of predominantly isozymes III, II, and I due to the instability of isozyme V during the hybridization procedure.The above results indicate that isozymes I and V are tetramers of the respective parental subunits and that isozymes II, III, and IV are hybrid molecules with subunit assignments of (I₃V₁), I₂V₂), and (I₁V₃), respectively.     

Selective production of L-aspartic acid and L-phenylalanine by coupling reactions of aspartase and aminotransferase in Escherichia coli

With L-aspartate (L-Asp) as the amino donor, L-phenylalanine (L-Phe) can be prepared from phenylpyruvate (PPA) via an amination reaction mediated by aminotransferase (encoded by aspC). On the other hand, L-Asp can be produced by an aspartase (encoded by aspA) -catalyzed reaction using fumaric acid as substrate. To overproduce aspartase in Escherichia coli, the aspA gene was cloned and overexpressed 180 times over the wild-type level. The use of AspA-overproducing E. coli strain for L-Asp production exhibited an 83% conversion, approaching to the theoretical yield, whereas the wild-type strain obtained scarcely L-Asp. Furthermore, the recombinant strain overproducing both AspA and AspC was able to produce L-Asp and L-Phe simultaneously by using fumaric acid and PPA as substrates. As a result, the conversion yields obtained for L-Asp and L-Phe were 78% and 85%, respectively. In sharp contrast, the wild-type strain attained a conversion of L-Phe less than 15% and an undetectable level of L-Asp. This result illustrates a potential and attractive process to yield both L-Asp and L-Phe by coupling AspA and AspC. A further study on the repeated use of the recombinant strain immobilized with calcium alginate showed that after eight batch runs L-Asp conversion maintained roughly constant (around 75%), whereas L-Phe conversion dropped to 65% from 81%. This result indicates the stability of AspA being superior to AspC.     

Inactivation of G1ucose-6-Phosphate Dehydrogenase Isozymes from Human and Pig Brain by Aluminum

Prolonged intake of low levels of aluminum from the drinking water has been found to increase the aluminum content in rat brain homogenates and to reduce the activity of hexokinase and glucose-6-phosphate dehydrogenase (G6PD). To determine the interaction of G6PD with aluminum in the brain, we have recently purified two isozymes of G6PD (isozymes I and II) from human and pig brain. Unlike isozyme I, isozyme II also had 6-phosphogluconate dehydrogenase (6-PGD) activity. We report here that G6PD isozymes I and II from human and pig brain purified to apparent homogeneity are inactivated by aluminum. Aluminum did not affect the 6-PGD activity of isozyme II. The aluminum-inactivated enzyme contained 1 mol of aluminum/mol of enzyme subunit. The protein-bound metal ion was not dissociated by exhaustive dialysis at 4 degrees C against 10 mM Tris-HCl (pH 7.0) containing 0.2 mM EDTA. Preincubation of aluminum with citrate, NADP+, EDTA, NaF, ATP, and apotransferrin protected the G6PD isozymes against aluminum inactivation. However, when the G6PD isozymes were completely inactivated by aluminum, only citrate, NaF, and apotransferrin restored the enzyme activity. The dissociation constants for the enzyme-aluminum complex of the isozymes varied from 2 to 4 microM, as measured by using NaF, a known chelator for aluminum. Inhibition of G6PD by low levels of aluminum further strengthens the suggested role of aluminum toxicity in the energy metabolism of the brain.     

In vitro inhibitory effects of some heavy metals on human erythrocyte carbonic anhydrases

The inhibition of two human carbonic anhydrase (HCA, EC 4.2.1.1) isozymes, the cytosolic HCA I and II, with heavy metal salts of Pb(II), Co(II) and Hg(II)has been investigated. Human erythrocyte CA-I isozyme was purified with a specific activity of 920 EUmg^{? 1} and a yield of 30% and CA-II isozyme was purified with a specific activity of 8000 EUmg^{? 1} and a yield of 40% using Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. The overall purification was approximately 104-fold for HCA-I and 900-fold for HCA-II. The inhibitory effects of different heavy metals (lead, cobalt and mercury) on CA activity were determined at low concentrations using the esterase method under in vitro conditions. K_i values for these metals were calculated from Lineweaver-Burk graphs as 1.0, 3.22 and 1.45 mM for HCA-I and 0.059, 1.382 and 0.32 mM for HCA-II respectively. Lead was a noncompetitive inhibitor for HCA-I and competitive for HCA-II, cobalt was competitive for HCA-I and noncompetitive for HCA-II and mercury was uncompetitive for both HCA-I and HCA-II. Lead was the best inhibitor for both HCA-I and HCA-II.     

Metabolism of d,l-chloro-phenylalanines by phenylalanine aminotransferase isozymes purified from bushbean shoots

A series of mono-, di- and trichloro-d,l-phenylalanines was tested as substrates for both phenylalanine aminotransferase and phenylalanine decarboxylase partially purified from bushbean (Phaseolus vulgaris L.) seedling extracts by ammonium sulphate fractionation and Sephacryl S-300 gel filtration. While most of the d,l-chlorophenylalanines were transaminated at rates of 35-100% of that observed with d,l-phenylalanine, no chlorophenylalanine decarboxylase activity was observed. A transamination reaction is therefore likely to be the initial step in the conversion of chloro-phenylalanines to their corresponding chloro-phenylacetic acids via a reaction pathway similar to the known route for the metabolism of l-phenylalanine to phenylacetic acid. The highest specific activity of phenylalanine aminotransferase was found in both root and shoot tissues of bushbean at the 10-day stage of seedling growth. Partially purified extracts of these tissues were able to transaminate most of the mono- and dichlorophenylalanines at ca 20-40% of the rate observed with d,l-phenylalanine, while the trichloro-phenylalanines (assayed at lower concentrations due to solubility) were transaminated at rates equal to those observed with d,l-phenylalanine. The 4-chloro derivative was the best substrate tested showing rates of transamination that were 25 % higher than those observed with d,l-phenylalanine. Further purification of shoot fractions by DEAE-Sephacel chromatography resolved the phenylalanine aminotransferase activity into two peaks (enzymes I and II) which on further purification, were found to behave differently during hydrophobic chromatography and PAGE. These results indicated the presence of two isozymic forms of phenylalanine aminotransferase in bushbean shoots and both were found to catalyse transamination of the monochloro-phenylalanines examined in this study.     

Purification and some properties of two NADP+-specific isocitrate dehydrogenases from an obligately psychrophilic marine bacterium, Vibrio sp., strain ABE-1.

Two isozymes of NADP+-specific isocitrate dehydrogenase [ICDH; EC 1.1.1.42] were confirmed to be present in an obligately psychrophilic marine bacterium, Vibrio sp., strain ABE-1, on the basis of the temperature-activity curve and electrophoretic mobilities. These isozymes were separated and purified about 170-fold for isozyme I (specific activity at 40 degrees C, 24.3 units/mg protein) and about 180-fold for isozyme II (specific activity at 20 degrees C, 59.2 units/mg protein), though the isozymes were still not homogeneous. The molecular weights of these isozymes determined by gel filtration were both about 85,000, but the properties of the isozymes were considerably different from each other. The thermostability of isozyme I resembled those of mesophiles, but isozyme II was extremely labile above 20 degrees C. NaCl affected the ICDH isozymes in different ways; the salt protected isozyme I from heat inactivation, but not isozyme II. Nevertheless it enormously enhanced the activity of isozyme II at low concentrations. Moreover, these ICDH isozymes showed different pH optima, Km values for isocitrate, susceptibilities to concerted inhibition by glyoxylate plus oxalacetate, and effects of 2-mercaptoethanol on their stabilities.     

Human T lymphocyte cAMP-dependent protein kinase: subcellular distributions and activity ranges of type I and type II isozymes.

The role of the type I and type II protein kinase A isozymes in the regulation of human T lymphocyte immune effector functions has not been ascertained. To approach this question, we first characterized the distribution and enzyme activities of the type I and type II protein kinase A (PKA) isozymes in normal, human T lymphocytes. T cells possess both type I and type II isozymes with an activity ratio of 5.0:1 +/- 0.71 (mean +/- SD). The type I isozyme associates predominately with the plasma membrane whereas the type II isozyme localizes primarily to the cytosol. Analyses of isozyme activities demonstrated that T cells from approximately one-third of 16 healthy donors exhibited significantly higher type II isozyme activities (higher type II, type IIH) than the remaining donors (lower type II, type IIL) (mean = 605 +/- 75 pmol.min-1.mg protein-1, P less than 0.001). Scatchard analyses of [3H]cAMP binding in the cytosolic fraction demonstrated similar Kd values (type IIH, 1.1 x 10(-7) M; type IIL, 9.0 x 10(-8) M); however, the Bmax (maximal binding) of the type IIH was 400 fmol/mg protein compared to the Bmax of the type IIL of 126 fmol/mg protein. Scatchard analysis of [3H]cAMP binding to the type I isozyme associated with membrane fragments had a Kd of 5.6 x 10(-8) M and a Bmax of 283 fmol/mg protein. Eadie-Hofstee plots of type IIH and type IIL gave a Km and Vmax of 2.3 mg/ml and 1.5 nmol.mg-1.min-1, and 2.1 mg/ml and 1.6 nmol.mg-1.min-1, respectively. The 3.2-fold higher maximal binding of the type II isozyme in one-third of healthy donors may reflect a greater amount of isozyme protein. The compartmentalization of type I PKA isozyme to the plasma membrane and type II PKA isozyme to the cytosol may serve to localize the isozymes to their respective substrates in T lymphocytes.     

Electrophoretic and biochemical studies of human aldehyde dehydrogenase isozymes in various tissues

Four major ALDH isozymes have been identified in human tissues using starch gel electrophoresis and isoelectric focusing. The isozyme bands have been termed as ALDH I, II, III and IV according to their decreasing electrophoretic migration and increasing isoelectric point. The isozymes have been partially purified via preparative isoelectric focusing. Kinetic characteristics of ALDH I and II were found to be quite similar to ALDH enzyme 2 and enzyme 1 described earlier by Greenfield and Pietruszko (Biochem Biophys Acta, $\text{483}$ 35–45 1977). ALDH III and IV showed a very high Km for propionaldehyde (1.0–1.5 mM at pH 9.5) and were not inhibited by disulfiram at pH 9.5. A variant phenotype of ALDH which lacked in isozyme I was detected in various tissues from Japanese individuals. Comparative kinetic properties of normal and variant enzyme are given.     

Secretion of Hexosaminidase Isozymes by Tetrahymena*

SYNOPSIS. Tetrahymena pyriformis strain HSM secrete large quantities of lysosomal acid hydrolases into the medium. The finding that 2 isozymes of β-N-acetylhexosaminidase (2-acetamido-2-deoxy-β-D-glucoside acetamidodeoxyglucohydrolase; EC 3.2.1.30) could be resolved by DEAE ion exchange chromatography and of possible differences between the secreted mixture and the intralysosomal hexosaminidase activity suggested that Tetrahymena might prove useful for studies of the control of lysosomal hydrolase isozyme secretion. In the present paper, we report a considerable purification of these isozymes and describe a number of their kinetic properties. Four isozymes were isolated into 2 major forms, A₁ and B₁, and 2 minor forms, A₂ and B₂, which were similar to the respective major forms in all kinetic properties tested. Hexosaminidase B₁ has a molecular weight of ?93,000 daltons and is inhibited by high concentrations of p-nitrophenyl-N-acetyl-β-D-glucosaminide. The inhibition is reversed by ethanol. Hexosaminidase A₁ has a molecular weight of ?170,000 and is not inhibited by high concentrations of substrate. The A forms are relatively less active against p-nitrophenyl-N-acetyl-β-D-galactosaminide than the B forms. Neither hexosaminidases A₁ or B₁ has any endo-β-N-acetylhexosaminidase activity. Comparison of the properties of the 2 major isozymes suggested that measurements of activity obtained under different assay conditions could be used to quantitate the amount of each isozyme in a mixture of the two. Log- and early stationary-phase cells secrete ?20% of isozyme A and 80% of isozyme B into the medium or into a dilute salt solution. With increasing culture age the fraction of isozyme A secreted rises to over 90%. Supplementation of the proteose-peptone growth medium with glucose causes a decrease in total hexosaminidase subsequently secreted but with no change in proportion of each isozyme. Cells suspended in a dilute salt solution containing 0.1 mM L-propranolol secrete slightly more isozyme A than do control cells suspended without L-propranolol. Phenoxy-benzamine (0.2 mM) causes a slight decrease in the proportion of isozyme A released.     

Two glyceraldehyde-3-phosphate dehydrogenase isozymes from the koningic acid (heptelidic acid) producer Trichoderma koningii

The sesquiterpene lactone koningic acid (heptelidic acid) irreversibly inactivated glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde 3-phosphate: NAD+ oxidoreductase (phosphorylating)] (EC 1.2.1.12) (GAPDH) and thus inhibits glycolysis. The koningic-acid-producing strain of Trichoderma koningii M3947 was shown to contain the koningic-acid-resistant GAPDH isozyme (GAPDH I) under conditions of koningic acid production. In peptone-rich medium, however, no koningic acid production was observed, and the koningic-acid-sensitive GAPDH isozyme (GAPDH II), in addition to the resistant enzyme, was produced. Both enzymes were tetramer with a molecular mass of 152 kDa (4 x 38 kDa) and lost enzyme activity when two of the four cysteine residues reacted with koningic acid. The apparent Km values of GAPDH I and II for glyceraldehyde 3-phosphate were 0.54 mM and 0.33 mM, respectively. The former isozyme was inhibited 50% by 1 mM koningic acid but not affected at 0.1 mM, while the latter isozyme was inhibited 50% at 0.01 mM. The immunochemical properties and partial amino acid sequences suggested that the two isozymes have different molecular structures. These results suggest that GAPDH I is responsible for the glycolysis in T. koningii when koningic acid is produced.     

Characterization of the esterase isozymes of Ips typographus (coleoptera,scolytidae)

Four esterase isozymes hydrolyzing α-naphthyl acetate (α-NA) were detected screening whole body homogenates of larvae and adults of Ips typographus by electrophoresis. Two of the four isozymes (isozymes 3 and 4) were not detected by α-NA staining in the pupal stage, but topical application of juvenile hormone III (JH III) on the pupa induced these isozymes. The JH esterase (JHE) activity on the gel was associated with the proteins of isozyme 2. The compounds OTFP, PTFP, and DFP inhibited this catalytic activity of isozyme 2 on the gel at low concentrations, whereas the proteins of isozyme 3 and 4 were affected only at higher concentrations. A quantitative developmental study was performed to characterize which of the esterases hydrolyzed JH III, using a putative surrogate substrate for JH (HEXTAT) and α-NA. The I₅₀ of several esterase inhibitors and the JH metabolites were also defined. All findings supported the results that a protein associated with isozyme 2 is catabolizing JH and that isozymes 3 and 4 are the main contributors to the general esterase activity on α-NA. The JHE from Tenebrio molitor was purified by affinity chromatography. Although the recovery was low, an analytical isoelectric focusing gel showed that the JHE activity of the purified enzyme. T. molitor cochromatographed at the same pl as the JHE activity of I. typographus. Arch. Insect Biochem. Physiol. 34:203–221, 1997. © 1997 Wiley-Liss, Inc.     

Changes in the isozyme composition of antioxidant enzymes in response to aminotriazole in leaves ofArabidopsis thaliana

The changes in isozyme profiles of catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and glutathione reductase (GR) during severe deactivation of total CAT activity by aminotriazole (AT) treatment were investigated in the leaves ofArabidopsis thaliana (Columbia ecotype) in relation to H₂O₂-mediated oxidative stress. In spite of striking deactivation of total CAT activity by 0.1 mM AT, there were no significant differences in H₂O₂ levels or total leaf soluble protein contents including a Rubisco in both the control and AT-treated leaves. On the other hand, one specific protein band (molecular mass, 66 kD) was observed on the SDS-gel from leaf soluble proteins whose staining intensity was strikingly enhanced by AT treatment for 6 h. However, this band disappeared at 12 h. In the native-gel assays of CAT, POD, APX and GR isozymes, AT remarkably inhibited the expression of the CAT1 isozyme with no effects on CAT2 and CAT3, and generally had no effect on POD isozyme profiles. However, AT stimulated the intensity of activities of pre-existing APX1 and GR1 isozymes. In particular, it induced a new synthesis of one GR isozyme. Therefore, these results collectively suggest that a striking deactivation of total CAT activity by AT inA. thaliana leaves largely results from the suppression of CAT1 isozyme, and that APX1, GR1, and a newly synthesized GR isozyme could complement the role of CAT1 to metabolize H₂O₂ into non-toxic water.     

Carbonic anhydrase inhibitors. Interaction of isozymes I, II, IV, V, and IX with organic phosphates and phosphonates

The interaction of five human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes, that is, hCA I, II, IV, V, and IX with a small library of phosphonic acids/organic phosphates, including methylphosphonic acid, MPA; phenylphosphonic acid, PPA; N-(phosphonoacetyl)-L-aspartic acid, PALA, methylene diphosphonic acid MDPA, the O-phosphates of serine (Ser-OP) and threonine (Thr-OP) as well as the antiviral phosphonate foscarnet has been studied. hCA I was activated by all these compounds, with the best activators being MPA and PPA (K(A)s of 0.10-1.20 microM). MPA and PPA were on the other hand nanomolar inhibitors of hCA II (K(I)s of 98-99 nM). PALA showed an affinity of 7.8 microM, whereas the other compounds were weak, millimolar inhibitors of this isozyme. The best hCA IV inhibitors were PALA (79 nM) and PPA (5.4 microM), whereas the other compounds showed K(I)s in the range of 0.31-5.34 mM. The mitochondrial isozyme was weakly inhibited by all these compounds (K(I)s in the range of 0.09-41.7 mM), similarly to the transmembrane, tumor-associated isozyme (K(I)s in the range of 0.86-2.25 mM). Thus, phosphonates may lead to CA inhibitors with selectivity against two physiologically relevant isozymes, the cytosolic hCA II or the membrane-bound hCA IV.     

Methylglyoxal as substrate and inhibitor of human aldehyde dehydrogenase: Comparison of kinetic properties among the three isozymes

Methylglyoxal was demonstrated to be a substrate for the isozymes E1, E2 and E3 of human aldehyde dehydrogenase. Pyruvate was the product from the oxidation of methylglyoxal by the three isozymes. At pH 7.4 and 25^oC, the major and minor components of the E3 isozyme catalyzed the reaction with V_max of 1.1 and 0.8 μmol NADH min⁻¹ mg⁻¹ protein, respectively, compared to 0.067 and 0.060 μmol NADH min⁻¹ mg⁻¹ protein for the E1 and E2 isozymes, respectively. The E2 isozyme had a K_m for methylglyoxal of 8.6 μM, the lowest compared to 46 μM for E1 and 586 and 552 μM for the major and minor components of the E3 isozyme, respectively. Both components of the E3 isozyme showed substrate inhibition by methylglyoxal, with K_i values of 2.0 mM for the major component and 12 mM for the minor component at pH 9.0. Substrate inhibition by methylglyoxal was not observed with the E1 and E2 isozymes. Methylglyoxal strongly inhibited the glycolaldehyde activity of the E1 and E2 isozymes. Mixed-type models of inhibition were employed as an approach to calculate the inhibition constants, 44 and 10.6 μM for E1 and E2 isozymes, respectively.     

Carbonic anhydrase inhibitors. Inhibition of isozymes I, II, IV, V, and IX with anions isosteric and isoelectronic with sulfate, nitrate, and carbonate

The inhibition of five human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes; the cytosolic hCA I and II, the membrane-bound hCA IV, the mitochondrial hCA V, and the tumor-associated, transmembrane hCA IX, with anions isosteric and isoelectronic with sulfate, nitrate, and carbonate; such as chlorate, perchlorate, bromate, iodate, periodate, silicate, bismuthate, vanadate, molybdate, and wolframate is reported. Apparently, the geometry of the inhibitor (tetrahedral or trigonal) does not influence its binding to the Zn(II) ion of the enzyme active site, but the nature of the central element is the most important factor influencing potency. Isozymes hCA I and II are best inhibited by chlorate, perchlorate, and silicate, together with the anions structurally related to sulfate, sulfamate, and sulfamidate, but sulfate itself is a weak inhibitor (inhibition constant of 74 mM against hCA I and 183 mM against hCA II). Molybdate is a very weak hCA I inhibitor (K(I) of 914 mM) but it interacts with hCA II (K(I) of 27.5mM). Isozyme IV is well inhibited by sulfate (K(I) of 9 mM), sulfamate, and sulfamidate (in the low micromolar range), but not by perchlorate (K(I) of 767 mM). The mitochondrial isozyme V has the lowest affinity for sulfate (K(I) of 680 mM) and carbonate (K(I) of 95 mM) among all the investigated isozymes, suggesting on one hand its possible participation in metabolon(s) with sulfate anion exchanger(s), and on the other hand an evolutionary adaptation to working at higher pH values (around 8.5 in mitochondria) where rather high amounts of carbonate in equilibrium with bicarbonate may be present. Metasilicate, isosteric to carbonate, is also about a 10 times weaker inhibitor of this isozyme as compared to other CAs investigated here (K(I) of 28.2 mM). Surprisingly, the tumor-associated isozyme IX is resistant to sulfate inhibition (K(I) of 154 mM) but has affinity in the low micromolar range for carbonate, sulfamate, and sulfamidate (K(I) in the range of 8.6-9.6 microM). This constitutes another proof that this isozyme best works at acidic pH values present in tumors, being inhibited substantially at higher pH values when more carbonate may be present. Bromate and chlorate are quite weak CA IX inhibitors (K(I) s of 147-274 mM).     

Carbonic anhydrase inhibitors. Interaction of isozymes I, II, IV, V, and IX with carboxylates

A detailed inhibition study of five carbonic anhydrase (CA, EC 4.2.1.1) isozymes with carboxylates including aliphatic (formate, acetate), dicarboxylic (oxalate, malonate), hydroxy/keto acids (l-lactate, l-malate, pyruvate), tricarboxylic (citrate), or aromatic (benzoate, tetrafluorobenzoate) representatives, some of which are important intermediates in the Krebs cycle, is presented. The cytosolic isozyme hCA I was strongly activated by acetate, oxalate, pyruvate, l-lactate, and citrate (K(A) around 0.1 microM), whereas formate, malonate, malate, and benzoate were weaker activators (K(A) in the range 0.1-1mM). The cytosolic isozyme hCA II was weakly inhibited by all the investigated anions, with inhibition constants in the range of 0.03-24 mM. The membrane-associated isozyme hCA IV was the most sensitive to inhibition by carboxylates, showing a K(I) of 99 nM for citrate and oxalate, of 2.8 microM for malonate and of 14.5 microM for pyruvate among others. The mitochondrial isozyme hCA V was weakly inhibited by all these carboxylates (K(I)s in the range of 1.67-25.9 mM), with the best inhibitor being citrate (K(I) of 1.67 mM), whereas this is the most resistant CA isozyme to pyruvate inhibition (K(I) of 5.5mM), which may be another proof that CA V is the isozyme involved in the transfer of acetyl groups from the mitochondrion to the cytosol for the provision of substrate(s) for de novo lipogenesis. Furthermore, the relative resistance of CA V to inhibition by pyruvate may be an evolutionary adaptation of this mitochondrial isozyme to the presence of high concentrations of this anion within this organelle. The transmembrane, tumor-associated isozyme hCA IX was similar to isozyme II in its slight inhibition by all these anions (K(I) in the range of 1.12-7.42 mM), except acetate, lactate, and benzoate, which showed a K(I)>150 mM. The lactate insensitivity of CA IX also represents an interesting finding, since it is presumed that this isozyme evolved in such a way as to show a high catalytic activity in hypoxic tumors rich in lactate, and suggests a possible metabolon in which CA IX participates together with the monocarboxylate/H(+) co-transporter in dealing with the high amounts of lactate/H(+) present in tumors.     

Partial purification and characterization of L-lactate dehydrogenase isozymes from sweet potato roots.

Lactate dehydrogenase [L-lactate: NAD oxidoreductase, EC 1.1.1.27] was isolated from sweet potato root tissues. Two species of the enzyme (isozymes I and II) were separated by DE-52 cellulose column chromatography from healthy, cut, and black-rot diseased tissues. Isozymes I and II were purified from healthy and diseased tissues, respectively. Reduction of pyruvate by NADH with either isozyme I or II was inhibited by pyruvate at high concentrations, by NAD+ and by several mononucleotides. Isozyme I was inhibited by a lower concentration of adenine nucleotide than isozyme II, and Km for pyruvate was increased markedly at acidic pH in the case of isozyme I, but only slightly in the case of isozyme II. The molecular weights of both isozymes were determined to be 150,000 and they were found to be charge isomers by polyacrylamide gel electrophoresis. The enzyme activity increased in response to infection by black-rot fungus but decreased in response to cutting.     

Carbonic anhydrase inhibitors: inhibition of the membrane-bound human isozyme IV with anions

The membrane-associated human isozyme of carbonic anhydrase, hCA IV, has been investigated for its interaction with anion inhibitors, for the CO(2) hydration reaction catalyzed by this enzyme. Surprisingly, halides were observed to act as potent hCA IV inhibitors, with inhibition constants in the range of 70-90 microM, although most of these ions, and especially fluoride, the best hCA IV inhibitor among the halides, are weak inhibitors of other isozymes, such as hCA I, II and V. The metal poisons cyanate, cyanide and hydrogen sulfide were weaker hCA IV inhibitors (K(i)'s in the range of 0.6-3.9 mM), whereas thiocyanate, azide, nitrate and nitrite showed even weaker inhibitory properties (K(i)'s in the range of 30.8-65.1 mM). Sulfate was a good hCA IV inhibitor (K(i) of 9 mM), although it is a much weaker inhibitor of isozymes I, II, V and IX. Excellent hCA IV inhibitory properties showed sulfamic acid, sulfamide, phenylboronic acid and phenylarsonic acid, with K(i)'s in the range of 0.87-0.93 microM, whereas their affinities for the other investigated isozymes were in the millimolar range. The interaction of some anions with the mitochondrial isozyme hCA V has also been investigated for the first time here. It has been observed that among all these isozymes, hCA V has the lowest affinity for bicarbonate and carbonate (K(i)'s in the range of 82-95 mM), which may represent an evolutionary adaptation of this isozyme to the rather alkaline environment (pH 8.5) within the mitochondria, where hCA V plays important functions in some biosynthetic reactions involving carboxylating enzymes (pyruvate carboxylase and acetyl coenzyme A carboxylase). There are important differences of affinity for anions between the two membrane-associated isozymes, hCA IV and hCA IX.