Glomerular compromise in mercuric chloride-induced nephrotoxicity.

We have examined the effects of mercuric chloride on renal glomerular structure. Isolated glomeruli from mercury-treated rats (HgCl2, 5 mg/kg body wt, s.c.) 1 hour post injection presented a diminished cross-sectional area as compared with control glomeruli [control (micron2) = 26,310 +/- 2,545, HgCl2 (micron2) = 18,474 +/- 1,828] and higher glomerular calcium content (control = 23 +/- 6 nmoles/mg prot, HgCl2 = 43 +/- 7 nmoles/mg prot). Renal sections prepared for immunohistochemical and histochemical analysis showed larger deposits of fibronectin and lipids and enhanced cellularity in glomerular structures from HgCl2-treated rats. Moreover, mieloperoxidase activity measured in isolated glomeruli were also increased as compared with control preparations [MPO (U/mg prot): control = 59 +/- 7, HgCl2 = 134 +/- 10]. When the animals were studied 24 hours post HgCl2 injection, glomerular cross-sectional area values were not different from control values (25,276 +/- 1,983 micron2), while calcium contents were higher than values observed 1 hour after treatment (92 +/- 9 nmoles/mg prot). A similar pattern was observed in fibronectin deposits. Hypercellularity in glomerular structures and the higher mieloperoxidase levels were maintained at this time (MPO HgCl2-rats 24 h = 148 +/- 31 U/mg prot). The effects observed in this study are consistent with an inflammatory response in the glomerular structure of HgCl2-treated rats that could explain the altered renal function described in previous reports in our laboratory.

Effects of divalent cations and nucleotides on the 14CO2-oxaloacetate exchange catalyzed by the phosphoenol pyruvate carboxykinase from the moderate halophile, Vibrio costicola.

The phosphoenolpyruvate carboxykinase (PEPCK) from Vibrio costicola catalyzed a 14CO2-oxaloacetate exchange reaction with an unusual nucleotide specificity. ATP gave the higher apparent catalytic efficiency (Vmax/Km, 6.78), followed by GTP (1.30), CTP (0.87) and ITP (0.66). Maximal activity required a divalent cation; CdCl2 and MgCl2 synergistically activated the enzyme, when added in the presence of MnCl2. The sigmoidal saturation curve for MnCl2 (apparent n 2.11) was converted into a hyperbola by 0.01 mM CdCl2 (apparent n 1). The results suggest a double role of the divalent cation in the reaction mechanism, namely as part of the MeATP2- substrate and as free Me2+. Mn2+ would be the best for the first, and Cd2+ for the second role. Preincubation with 0.01 mM CdCl2 increased the activity of the enzyme assayed with MgATP2- through an increase in Vmax; addition of CdCl2 to the reaction mixture elicited further activation, through a 17-fold decrease in the apparent Km for MgATP2-. These results, together with the biphasic curve of activation by CdCl2 when used alone, suggest the existence of two different sites for free Cd2+ on the enzyme.

Phosphoenolpyruvate carboxykinase from the moderate halophile Vibrio costicola. Purification, physicochemical properties and the effect of univalent-cation salts.

Phosphoenolpyruvate carboxykinase (PEPCK) was purified to homogeneity from the moderately halophilic bacterium Vibrio costicola. The enzyme is monomeric, with an Mr of 62,000, as determined by the Svedberg equation, by using values of s0(20,w) 4.4 x 10(-13) s, D20,w 6.13 x 10(-7) cm2.s-1 and v 0.719 cm3.g-1. Compared with other, non-halophilic, PEPCKs, the enzyme from V. costicola had a significantly lower total content of hydrophobic amino acids. The contents of glycine and serine were higher in the V. costicola enzyme (16.7 and 10.22% respectively) than in the non-halophilic PEPCKs (6.8-9.6% and 4.67-6.28% respectively). These results resemble those obtained by De Médicis & Rossignol [(1979) Experientia 35, 1546-1547] with the pyruvate kinase from V. costicola, and agree with the proposal by Lanyi [(1974) Bacteriol. Rev. 38, 272-290] of partial replacement of hydrophobic amino acids by glycine and serine to maintain the balance between hydrophobic and hydrophilic forces in halophilic enzymes. In agreement with this 'halophilic' characteristic, the PEPCK was somewhat stabilized by 1 M-KCl or -NaCl and by 20% (v/v) glycerol, and its oxaloacetate-decarboxylation and 14CO2-oxaloacetate-exchange reactions were activated by KCl and NaCl up to 1 M, whereas the fixation of CO2 on PEP had a maximum at 0.025-0.05 M salt. These facts suggest that the salts, at concentrations probably physiological for the bacterium, increase the formation of the complex of oxaloacetate and ATP with the enzyme, and the liberation of the products, PEP and ADP, thus favouring PEP synthesis.

Citrate synthase from Crithidia fasciculata: inhibition by adenine nucleotides and suramin.

Citrate synthase (EC 4.1.3.7) was purified to electrophoretic homogeneity from Crithidia fasciculata ATCC 11745. 2. The purified enzyme had an optimal pH of 8.0-8.5, apparent Km values for acetyl-CoA and oxaloacetate of 5.5 and 3.5 microM, respectively, and was not activated by NH4Cl or KCl, nor inhibited by NADH or alpha-oxoglutarate. 3. Adenine nucleotides inhibited the enzyme, ATP being the most effective. The inhibition was strictly competitive towards acetyl-CoA and of the mixed type with respect to oxaloacetate. 4. The trypanocidal drug suramin inhibited both the C. fasciculata and the pig liver citrate synthases, being strictly competitive with respect to oxaloacetate, and non-competitive towards acetyl-CoA. The competitive inhibition with respect to the divalent anion oxaloacetate might be due to the strongly anionic nature of suramin, which has six sulfonic groups in its molecule.

Some properties of the NADP-specific malic enzyme from the moderate halophile Vibrio costicola.

NADP-specific malic enzyme (EC 1.1.1.40) has been purified about 160-fold from the moderate halophile Vibrio costicola. The enzyme has a molecular weight of about 120,000. The purified enzyme was unstable in dilute solutions but could be stabilised by NaCl or glycerol. NH4Cl or KCI caused maximal activation at 0.1M, but higher concentrations were inhibitory. NaCl did not activate and was instead a mixed-type inhibitor towards NH4Cl or KCI. The salt concentration affected the kinetic parameters of the reaction. The apparent Km for L-malate reached a minimal value at about 0.1 M salt; the value for NADP, on the other hand, increased continuosly with the Co2+ or Mg2+. NADH was a mixed-type inhibitor towards both substrates, whereas oxaloacetate was strictly competitive towards L-malate and non-competitive towards NADP. The inhibition kinetics were sigmoidal for NADH and hyperbolic for oxaloacetate. The malic enzyme form V. costicola was similar to those of a marine Pseudomonas and Halobacterium cutirubrum in kinetic and regulatory properties but showed a response to salts intermediate between those of the latter enzymes.