Inhibition of several enzymes by gold compounds. II. beta-Glucuronidase, acid phosphatase and L-malate dehydrogenase by sodium thiomalatoraurate (I), sodium thiosulfatoaurate (I) and thioglucosoaurate (I).

Bovine liver beta-D-glucuronide glucuronohydrolase, EC 3.2.1.32), wheat germ acid phosphatase (orthophosphoric monoesterphosphohydrolase, EC 3.1.3.2) and bovine liver L-malate dehydrogenase (L-malate: NAD oxidoreductase, EC 1.1.1.37) were inhibited by a series of gold (I) complexes that have been used as anti-inflammatory drugs. Both sodium thiosulfatoaurate (I) (Na AuTs) and sodium thiomalatoraurate (NaAuTM) effectively inhibited all three enzymes, while thioglucosoaurate (I) (AuTG) only inhibited L-malate dehydrogenase. The equilibrium constants (K1) ranged from nearly 4000 microM for the NaAuTM-beta-glucuronidase interaction to 24 microM for the NaAuTS-beta-glucuronidase interaction. The rate of covalent bond formation (kp) ranged from 0.00032 min-1 for NaAuTM-beta-glucuronidase formation to 1.7 min-1 for AuTG-L-malate dehydrogenase formation. The equilibrium data shows that the gold (I) drugs bind by several orders lower than the gold (III) compounds, suggesting a significantly stronger interaction between the more highly charged gold ion and the enzyme. Yet the rate of covalent bond formation depends as much on the structure of the active site as upon the lability of the gold-ligand bond. It was also observed that the more effective the gold inhibition the more toxic the compound.

Inhibition of hydrolytic enzymes by gold compounds. I. beta-Glucuronidase and acid phosphatase by sodium tetrachloroaurate (III) and potassium tetrabromoaurate (III).

Purified bovine liver beta-glucuronidase (beta-D-glucuronide glucuronohydrolase, EC 3.2.1.32) and wheat germ acid phosphatase (orthophosphoric monoesterphosphohydrolase, EC 3.1.3.2) were inhibited with freshly dissolved and 24 h aquated tetrahaloaurate (III) compounds. Rate and equilibrium inhibition constants were measured. From this data two acid phosphatases species were observed. Equilibrium inhibition constants ranged from 1 to 12.5 microM for the various gold compounds toward both enzymes. The first order rate constants ranged between 0.005 and 0.04 min.-1 for most reactions with the exception of the fast reacting acid phosphatase which had values as high as 2.6 and 2.8 min.-1. It is observed that the beta-glucuronidase is rapidly inhibited during the equilibrium phase before the more slower reaction covalent bond formation takes place. The acid phosphatases form the covalent bonds more rapidly, especially the faster reacting species suggesting a unique difference in the active site geometry to that of the more slowly reacting species. The tightly bonded gold (III)-enzyme complex is probably the reason for its toxicity and non-anti-inflammatory use as a drug.

Two calcium currents in a smooth muscle cell line.

Membrane currents in small cells of a smooth muscle cell line (A10) derived from embryonic rat thoracic aorta were monitored by the patch electrode whole-cell voltage clamp technique. Three currents, two divalent cation currents, and a Ca2+-activated K+ current have been observed. The latter is readily abolished pharmacologically, allowing the characterization of the divalent cation currents. With a holding potential of -50 mV, a single divalent current, which inactivates slowly, is elicited on depolarization of the membrane potential to values positive to ca. -10 mV. The second divalent cation current is only observed when the holding potential is negative to -55 mV and the membrane is pulsed to values positive to ca. -35 mV. This current is rapidly inactivating, peaking in approximately 5 ms and decaying with a t1/2 of ca. 15 ms at 0 mV when conveyed by Ba2+. The rapidly inactivating divalent cation current is depressed by substitution of Ba2+ for Ca2+ in the bathing solution and is highly insensitive to organic Ca2+ channel blockers. The slowly inactivating channel has more typical characteristics of Ca2+ channels; it is more permeable to Ba2+ than to Ca2+ and is sensitive to modulation by dihydropyridines. These data demonstrate the presence of two distinctly different Ca2+ channels in A10 cells.

Effects of cis- and trans-platinum complexes on RNA transcription.

Current thinking concerning the anti-cancer actions of platinum complexes view cis-platinum agents as active and trans-complexes as not being anti-neoplastic. It is generally accepted that the active cis forms exert their effects by binding to the DNA template. Data is presented here showing trans-Pt(NH3)2Cl2(trans-PDD) to be much more active than cis-Pt(NH3)2Cl2(cis-PDD) in inhibiting in vitro RNA transcription using both bacterial and eukaryotic enzyme sources. It is further shown that the polymerase enzyme is a much more sensitive target for these platinum agents than the DNA template under the conditions employed.

Inhibition of two mitochondrial enzymes by gold (III) halo complexes. Evidence for a binding mechanism.

Fumarase (EC 4.2.1.2) and mitochondrial L-malate dehydrogenase (EC 1.1.1.37) were both inhibited by NaAuCl4 and KAuBr4. The inhibition for both was measured as a function of gold complex concentration and aquation time, and the NaAuCl4 inhibition was also measured in the presence of 0.15 M NaCl. Regeneration of the enzyme activity after NaAuCl4 inhibition using L-cysteine, L-methionine and NaCN was also investigated. Sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis and amino acid analysis was performed on the NaAuCl4 inhibited enzymes as well as on ribonuclease A (EC 3.1.26.2), lysozyme (EC 3.2.1.17) and liver alcohol dehydrogenase (EC 1.1.1.1). It was observed that the inhibition was proportional to the gold complex concentration but decreased markedly after aquation of the complex. In the presence of NaCl the initial rate of inactivation is essentially unaffected unless the complex has been aquated and then the initial rate is increased. Gel electrophoresis on gold complex-enzyme mixtures show polymerization for ribonuclease and lysozyme and amino acid analysis indicates that no oxidation has taken place. From these results, a binding mechanism is postulated for the inhibition of the dehydrogenases by direct displacement of a halide ligand, probably by two groups on the enzyme, at least one of which may be a sulfur containing acid.

The effects of platinum complexes on seven enzymes.

The effects of K2PtCl4, cis-Pt(NH3)2Cl2, and trans-Pt(NH3)2Cl2 on the activities of glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, dihydrofolate reductase, fructose-1,6-bisphosphate aldolase, catalase, tyrosinase, and peroxidase have been investigated. All of the enzymes which are thought to have essential sulfhydryl groups (glyceraldehyde-3-phosphate dehydrogenase, aldolase, and glucose-6-phosphate dehydrogenase) were significantly inhibited by K2PtCl4. The other four enzymes studied are not known to have essential sulfhydryl groups, and were not significantly affected by the Pt compounds under the conditions employed. Glyceraldehyde-3-phosphate dehydrogenase was the only enzyme inhibited by all three Pt compounds tested, with K2PtCl4 being the most effective and cis-Pt(NH3)2Cl2 the least effective inhibitor. Semilogarithmic plots of residual activity versus inhibition time indicated that the inhibition reactions were not simple first-order processes, except for the inhibition of glucose-6-phosphate dehydrogenase by K2PtCl4 which appeared to be first-order with respect to enzyme concentration.

Enhanced inhibition of both cellular protein synthesis and malate dehydrogenase by aged aquoplatinum(II) complexes.

Previous studies have shown cis-diamminedichloroplatinum(II) (Cis) an effective anti-tumour agent in man and animals. Evidence is presented here that formation of aquo complexes of this platinum derivative will significantly enhance its inhibitory properties with respect to two separate biochemical functions, namely inhibition of protein synthesis in hamster medulloblastoma cells and in inhibiting the activity of L-malate dehydrogenase (MDH) in a cell free system. Inhibition of cell protein synthesis rises from 8% using freshly dissolved drug to 30% when aged solutions of drug are employed at an inhibitor concentration of 0.1 mM. The inhibitory enhancement seen using purified malic dehydrogenase increases from 16% (fresh) to 57% (aged) at an inhibitor concentration of 1 mM.

Implication of a tyrosyl residue at the active site of mitochondrial L-malate:NAD+ oxidoreductase.

The tyrosyl residues of porcine mitochondrial L-malate:NAD+ oxidoreductase (EC 1.1.1.37) have been studied spectrophotometrically and using selective chemical modification with iodine and tetranitromethane. CNBr hydrolysis and Sephadex G-25, G-50 and G-75 chromatography produced a peptide which contained two tyrosines in the native and the nitrated molecules when the nitration took place in an NAD+-oxaloacetate solution. Nitration in the absence of the substrates caused the tyrosyl residues to disappear. Spectrophotometric titrations indicate that one of the 10 tyrosyl residues in mitochondrial L-malate:NAD+ oxidoreductase titrate abnormally, while iodination experiments suggest that two fast-reacting tyrosines are not involved in activity. Nitration and iodination experiments, in conjunction with CNBr-mapping, suggest that two of the four nitrated tyrosyl residues are necessary for biological action. Titration of the sulfhydryl groups with 4,4-bis-dimethylaminodiphenyl carbinol before and after nitration indicate that none of the cysteinyl residues were oxidized by the tetranitromethane, thus ruling out the loss of enzyme activity due to the thiol oxidation.

Interactions of cis- and trans-platinum(II) complexes with dehydrogenase enzymes in the presence of different mono- and polynucleotides: evidence for a ternary complex.

The inhibition of several dehydrogenase enzymes by cis- and trans-Pt(NH3)2Cl2 have been measured in the presence of baker yeast ribonucleic acid (RNA), calf thymus and salmon sperm deoxyribonuclic acid (DNA) and several mononucleotides (AMP and ATP). The binding constants for the interaction of the platinum complexes to the nucleotides have been calculated and a comparison of those values to the previously calculated platinum complex-enzyme binding constants strongly suggest that platinum compounds are more tightly bound to the enzymes. The binding of the platinum complexes to most of the enzymes was decreased in the presence of any nucleotide, yet it was observed that when using rabbit muscle (M4) lactate dehydrogenase the mononucleotides reduced the binding to a lesser degree while the polynucleotides actually enhanced the platinum-enzyme interaction. The implications of these interactions are discussed.

A proposed mechanism relating the antitumor behavior of cis-platinum amine complexes to their inhibition of a model enzyme.

The twenty-four hour inhibition of m-malate dehydrogenase (E.C. 1.1.1.37) by various complexes of cis-platinum(II) and cis-platinum(IV) was measured as a function of the platinum concentration. It was observed that increased alkylation of the amine groups of Pt(II) and to a lesser degree of Pt(IV) decreased the activity consistently. It was also observed that the Pt(IV) analogues inhibit the enzyme to about an order of magnitude greater than the Pt(II) complexes. These phenomena will be interpreted.

Protection of the active site of mitochondrial malate dehydrogenase from inhibition by potassium tetrachloroplatinate.

Mitochondrial malate dehydrogenase (L-malate : NAD-+ oxido-reductase, EC 1.1.1.37) was inhibited by potassium tetrachloro platinum (II), K-2PtCl-4, in the presence of varying concentrations of NADH, NAD-+ and L-malate and mixtures of NAD-+ and L-malate. It was observed that NADH is an effective protector of the enzyme from inhibition while both NAD-+ and L-malate are poor protectors. Spectral studies have suggested that the protection afforded by the substrates are accomplished by reaction with specific groups on the enzyme rather than by complexation of the substrates with PtCl-4-2-minus. From the above data it has been concluded that the tetrachloroplatinate ion binds only at the active site and that this site which is effectively protected by NADH, and moderately protected by a NAD-+-L-malate complex probably contains one or more sulfur containing amino acid side chains. It is also proposed that when the tetrachloroplatinate complexes with the enzyme there is some effect, possibly a conformational change, which causes the release of NADH at the allosteric site.