Rat Kidney Enzyme Research Articles

Complete Amino Acid Sequence of Rat Kidney Ornithine Aminotransferase: Identity with Liver Ornithine Aminotransferase1

The complete amino acid sequence of rat kidney ornithine aminotransferase [EC 2.6.1.13] is presented. The 404-residue sequence was determined by analysis of peptides generated by digestion of the S-carboxyamidomethylated protein with CNBr, Achromobacter protease I, arginylendopeptidase, or Staphylococcus aureus V8 protease. Mueckler and Pitot have reported the amino acid sequence of the rat liver enzyme (440 residues) as predicted from the nucleotide sequence of the cDNA [Mueckler, M.M. & Pitot, H.C. (1985) J. Biol. Chem. 260, 12993-12997]. The amino acid sequence of the rat kidney enzyme presented herein coincides with residue 36 (Gly) through 440 (Phe) of the predicted precursor protein, indicating that the liver and kidney enzymes are identical, and that the enzyme is processed at the amino-terminal region after translation.

Prenatal Development of Antioxidant Enzymes in Rat Lung, Kidney, and Heart: Marked Increase in Immunoreactive Superoxide Dismutases, Glutathione Peroxidase, and Catalase in the Kidney

The immaturity of antioxidant capacity in the lung in preterm newborn infants is postulated to contribute to the development of hyperoxic lung injury. Antioxidant enzymes in fetal lung, comprised of copper-zinc (cytosolic) and manganese (mitochondrial) superoxide dismutases, glutathione peroxidase, and catalase, have been reported to increase during the late gestational period. To determine whether such maturation of antioxidant capacity occurs in other tissues, we have evaluated the development of these four enzymes from d 18 to 22 of gestation in rat lung, kidney, and heart. To resolve the confusion in the reported levels of lung superoxide dismutases, the two isoenzymes were assayed separately by specific RIA. The growth of the kidney exceeded that of the whole body during this period, while the growth of the lung and heart did not. The concentrations of the four antioxidant enzymes in lung and kidney increased in a stepwise manner during this period, and the magnitude of the change for each enzyme was greater in the kidney than in the lung. On the other hand, the only significant change in the concentrations of heart antioxidant enzymes observed was a mild increase in the glutathione peroxidase concentration from d 20 to 22. These results suggest that the prenatal maturation of antioxidant capacity occurs earlier in the heart and later in the kidney than in the lung, and that the immaturity of antioxidant capacity could make the fetal rat kidney vulnerable to free radical-mediated injury.

Angiotensin II Receptors in the Kidney

Angiotensin II (Ang II) exerts a variety of actions through specific receptor binding in the kidney. These include modulation of renal hemodynamics, glomerular filtration rate (GFR), and tubular reabsorption. Quantitative in vitro autoradiography was used to localize the Ang II receptors and angiotensin converting enzyme (ACE) in rat, rabbit, and human kidney. We incubated 20 microns cryostat sections of kidney with either 125I-[Sar1]Ang II or 125I-[Sar1,Ile8]Ang II for Ang II binding or with 125I-351A for ACE localization. The resulting autoradiographs were analyzed by computerized densitometry. A high density of Ang II binding was found over glomeruli and vasa recta bundles in all species examined. The interbundle area of the outer medulla was associated with low to moderate binding of Ang II. Moderate binding of Ang II was observed over proximal convoluted tubules in the outer cortex of rat kidney. ACE was predominantly distributed in proximal convoluted tubules in all species. These studies reveal multiple sites where Ang II could modulate renal function by acting on the renal vasculature, glomeruli, vasa recta bundles, and proximal tubular sites.

Thiol antidotes effect on lipid peroxidation in mercury-poisoned rats

The effect of thiol antidotes 2,3-dimercapto-1-propanesulfonic acid (DMPS) and d-penicillamine (PA) on lipid peroxidation and on activities of some protecting enzymes in blood, liver and kidneys of mercury-poisoned rats has been studied. It has been found that Hg-poisoning is associated with increased lipid peroxidation in the liver and in the kidneys and with inactivation of superoxide dismutase (SOD) and catalase. Inhibition of SOD is caused by thiols treatment too, but in this case acceleration of lipid peroxidation has not been observed. Evidence is presented that in the liver, protection against mercury-induced lipid peroxidation is afforded by both thiols, while in the kidneys only PA has protective effect. In in vitro experiments it has been demonstrated that both antidotes can act as O 2 − scavengers and lipid peroxidation inhibitors, but PA is significantly more effective. On the basis of the obtained results a conclusion is drawn that in addition to the metal-removing ability, the antioxidant properties of the chelating agents may play an important role in manifestation of their beneficial effect in metal intoxications.

Nutritional and hormonal regulation of mRNA abundance for arginine biosynthetic enzymes in kidney

Argininosuccinate synthetase and argininosuccinate lyase catalyze the synthesis of arginine from citrulline in kidney and also serve as components of the urea cycle in liver of ureotelic animals. Dietary and hormonal regulation of mRNAs encoding these enzymes have been well studied in liver but not in kidney. Messenger RNAs for these enzymes are localized within the renal cortex. Starvation and extreme variations in dietary protein content (0% vs 60% casein) produced 2.6- to 3.5-fold increases in mRNA abundance for these two enzymes in rat kidney. Argininosuccinate lyase mRNA was not induced by dibutyryl cAMP, dexamethasone, or a combination of the two agents. In contrast, argininosuccinate synthetase mRNA was induced 2-fold by dibutyryl cAMP but was unresponsive to dexamethasone. Thus, diet and hormones regulate levels of these mRNAs in rat kidney, but the responses are both qualitatively and quantitatively distinct from the responses previously reported for rat liver.

Purification and properties of 2-aminoadipate: 2-oxoglutarate aminotransferase from bovine kidney.

Previous studies with rat kidney preparations indicated that 2-aminoadipate aminotransferase (AadAT) and kynurenine aminotransferase (KAT) activities are properties of a single protein. We found that bovine kidney contains an appreciable amount of AadAT activity, but lacks KAT activity. AadAT from bovine and rat kidney extracts were purified to electrophoretic homogeneity. The purification procedure included fractionation with (NH1)2SO1, heat treatment, DEAE-cellulose chromatography and hydroxyapatite chromatography. Physical and kinetic properties, such as pH optima, Km for substrates, Mr, electrophoretic mobility and inhibition by dicarboxylic acids of bovine kidney AadAT, were similar to those of the rat kidney enzyme. However, bovine kidney AadAT differed from rat kidney AadAT in substrate specificity, amino acid composition and stability when stored. The titration curve of bovine kidney AadAT was also different from that of the rat kidney enzyme. The results suggest that bovine kidney AadAT may have some structural similarity to rat kidney AadAT and that the structural differences observed between the two enzymes may explain the absence of KAT activity in bovine kidney.

Degradation of atrial natriuretic peptides by an enzyme in rat kidney resembling neutral endopeptidase 24.11

The inactivation of rat atrial natriuretic factor (ANF) was studied using a bioassay, ANF-stimulated guanylate cyclase activity. Rat kidney membranes degraded ANF into biologically inactive forms. The primary cleavage site appears to be the Cys 105-Phe 106 bond. The degradation, measured by HPLC, followed classical Michaelis-Menten kinetics. The sensitivity of the enzyme to inhibitors suggested it to be a metalloendopeptidase, resembling neutral endopeptidase 24.11. When this enzyme, characterised by its enkephalin-degrading activity, was compared to the enzyme responsible for ANF inactivation, striking differences were found in tissue distribution, pH-dependence and sensitivity to protein-modifying reagents. It is concluded that an enzyme similar to endopeptidase 24.11 may be responsible for inactivation of atrial peptides in the rat.

The metabolism of neuropeptides. Hydrolysis of peptides by the phosphoramidon-insensitive rat kidney enzyme ‘endopeptidase-2’ and by rat microvillar membranes

Endopeptidase-2, the second endopeptidase in rat kidney brush border [Kenny & Ingram (1987) Biochem. J. 245, 515-524] has been further characterized in regard to its specificity and its contribution to the hydrolysis of peptides by microvillar membrane preparations. The peptide products were identified, after incubating luliberin, substance P, bradykinin and angiotensins I, II and III with the purified enzyme. The bonds hydrolysed were those involving a hydrophobic amino acid residue, but this residue could be located at either the P1 or P1' site. Luliberin was hydrolysed faster than other peptides tested, followed by substance P and bradykinin. Human alpha-atrial natriuretic peptide and the angiotensins were only slowly attacked. Oxytocin and [Arg8]vasopressin were not hydrolysed. No peptide fragments were detected on prolonged incubation with insulin, cytochrome c, ovalbumin and serum albumin. In comparison with pig endopeptidase-24.11 the rates for the susceptible peptides were, with the exception of luliberin, much lower for endopeptidase-2. Indeed, for bradykinin and substance P the ratio kcat./Km was two orders of magnitude lower. Since both endopeptidases are present in rat kidney microvilli, an assessment was made of the relative contributions to the hydrolysis of luliberin, bradykinin and substance P. Only for the first named was endopeptidase-2 the dominant enzyme; for bradykinin it made an equal, and for substance P a minor, contribution.

Purification of ornithine aminotransferase by immunoadsorption

Ornithine aminotransferase was purified by conventional biochemical methods from rat kidney, rat liver, and human liver. Affinity-purified antibodies raised to the rat kidney enzyme were used to produce an immunoadsorbent enabling a one-step purification of ornithine aminotransferase to be made from crude human liver extracts. The harsh chemical conditions often required to desorb immunoadsorbents were avoided by isolating antibodies with low functional affinity and employing an electrophoretic desorption method which allowed the enzyme activity to be retained. The close structural similarity between human and rat ornithine aminotransferase was demonstrated by immunodiffusion reactions. It was therefore possible to purify the enzyme from human liver using immobilized antibodies raised against rat kidney ornithine aminotransferase. Furthermore, desorption was more readily achieved due to the lower affinity for the human enzyme.

On the active site thiol of gamma-glutamylcysteine synthetase: relationships to catalysis, inhibition, and regulation.

gamma-Glutamylcysteine synthetase (glutamate-cysteine ligase; EC 6.3.2.2) was isolated from an Escherichia coli strain enriched in the gene for this enzyme by recombinant DNA techniques. The purified enzyme has a specific activity of 1860 units/mg and a molecular weight of 56,000. Comparison of the E. coli enzyme with the well-characterized rat kidney enzyme showed that these enzymes have similar catalytic properties (apparent Km values, substrate specificities, turnover numbers). Both enzymes are feedback-inhibited by glutathione but not by gamma-glutamyl-alpha-aminobutyrylglycine; the data indicate that glutathione binds not only at the glutamate binding site but also at a second site on the enzyme that interacts with the thiol moiety of glutathione but not with a methyl group. Both enzymes are inactivated by buthionine sulfoximine in the presence of ATP, suggesting a common gamma-glutamyl phosphate intermediate. However, unlike the rat kidney enzyme that has an active center thiol, the bacterial enzyme is insensitive to cystamine, gamma-methylene glutamate, and S-sulfo amino acids, indicating that it does not have an active site thiol. Thus, the rat kidney and E. coli enzymes share several catalytic features but differ in active site structure. If the active site thiol of the rat kidney enzyme is involved in catalysis, which seems likely, there would appear to be differences in the mechanisms of action of the two gamma-glutamylcysteine synthetases.

Identification of protease 3.4.24.11 as the major atrial natriuretic factor degrading enzyme in the rat kidney

We have identified a metalloendoprotease from rat kidney cortex that cleaves the cysteine-phenylalanine bond (Cys 7-Phe 8) within the 17 amino acid ring structure of atrial natriuretic factor (ANF). Cleavage at this site represents the major ANF degradative activity in rat kidney, and is inhibited by the known metalloendoprotease inhibitors, thiorphan, phosphoramidon and zincov with IC 50 values in the nanomolar range. Since these are specific inhibitors of protease 3.4.24.11, both protease 3.4.24.11 and ANF degrading activities were monitored during purification. Both activities copurified at each chromatographic step. Furthermore, purified protease 3.4.24.11 cleaved ANF specifically at the Cys 7-Phe 8 bond. It is concluded from this work that the major ANF degrading enzyme in rat kidney is protease 3.4.24.11.

Metal induction of heme oxygenase.

The experimental data that have been reviewed support the following conclusions regarding metal induction of microsomal heme oxygenase activity: 1. Induction of heme oxygenase in liver, kidney, and other organs of rodents is a nonspecific, toxic response to parenteral administration of numerous metal compounds. 2. The Co2+ and Cd2+ ions are especially potent for induction of heme oxygenase in rat liver; Sn2+, Ni2+, and As3+ are especially potent for induction of the enzyme in rat kidney; Hg2+ is especially potent for induction of the enzyme in rat adrenal. 3. Rat spleen, testis, and brain are relatively refractory to metal induction of heme oxygenase activity; in testicular microsomes from Cd2+-treated rats, heme oxygenase activity is markedly inhibited. 4. Metal induction of heme oxygenase requires de novo synthesis of mRNA and protein, based on 1) experiments with metabolic inhibitors (actinomycin D, puromycin, and cycloheximide) and 2) translation assays of heme oxygenase mRNA. 5. Heme oxygenase induction by metals is generally suppressed by treatments with SH compounds (for example, cysteine and glutathione) and enhanced by agents that deplete tissue SH levels (for example, diethyl maleate), suggesting that the induction mechanism may involve binding of metal ions to SH-containing regulatory molecules. 6. Administration of DDC exerts a pronounced synergistic effect on Ni2+ induction of heme oxygenase activity in rat tissues, attributable in part to enhanced cellular uptake of nickel. 7. Induction of heme oxygenase is not sustained during repeated daily treatments of rats with NiCl2, pointing to an adaptive mechanism for tolerance to the enzyme inducer. Investigations of metal induction of heme oxygenase activity have commonly involved sc or ip administration of the test compounds to rats. The paucity of studies in other species and the dearth of experiments with oral or respiratory exposures hinder extrapolations of the animal data to human environmental or occupational exposures to metal compounds.

Induction of lipid peroxidation by oxalate in experimental rat urolithiasis

The function of lipid peroxidation and the antiperoxidative enzymes of rat liver and kidney were studied in stone formation induced by intraperitoneal administration of sodium oxalate (7 mg/100 g body weight). The animals sacrificed 3 and 12 h after administration of sodium oxalate had higher level of malondialdehyde in liver and kidney than control animals. A significantly pronounced release of malondialdehyde was observed in treated liver and kidney homogenates when incubated with either ferrous sulphate or hydrogen peroxide compared to control liver and kidney. Superoxide dismutase activity was increased only in liver and not in kidney in treated animals compared to the control. A highly significant decrease in catalase activity was observed in both liver and kidney of treated animals.

Effect of sodium lauryl sulphate on rat liver and kidney enzymes: an in vitro study.

Effect of sodium lauryl sulphate on rat liver and kidney enzymes: an in vitro study.

Effect of aurothioglucose on glutathione and glutathione-metabolizing and related enzymes in rat liver and kidney

The antirheumatic drug aurothioglucose is an inhibitor of the selenoenzyme GSH peroxidase. During chrysotherapy, the decreased levels of erythrocyte GSH and serum sulfhydryls of rheumatoid arthritis patients are normalized concomitant with clinical efficacy. This investigation examined the in vivo and in vitro effect of gold(I) as aurothioglucose on enzymes related to the GSH redox cycle or metabolism. The enzymes measured were GSH peroxidase, GSSG reductase, γ-glutamyl transpeptidase, γ-glutamylcysteine synthetase, GSH S-transferase, GSH thiotransferase, glucose-6-phosphate dehydrogenase, superoxide dismutase and catalase. Rats were injected with 30 μmol aurothioglucose/kg body wt. daily for 7 days by intramuscular injection. GSH levels in aurothioglucose-treated rats were 68% higher in erythrocytes ( P < 0.005) and 45% higher in kidney ( P < 0.001) than in control rats. Treatment with aurothioglucose did not elevate plasma or liver GSH. The enzyme activities that were changed by aurothioglucose treatment were GSH peroxidase in kidney (41% decreased, P = 0.005) and liver (13% decreased, P < 0.05), γ-glutamyl transpeptidase in kidney (15% decreased, P < 0.05), and catalase in kidney (58% decreased, P < 0.001). Kidney glucose-6-phosphate dehydrogenase activity was increased 50% ( P < 0.005) and GSH S-transferase was increased 72% ( P < 0.001). In vitro the only liver enzymes inhibited more than 50% by concentrations of less than 50 μM aurothioglucose were GSH peroxidase (50% inhibited by 25 μM aurothioglucose) and GSH thiotransferase (50% inhibited by 5 μM aurothioglucose). Studies of in vitro enzyme inhibition by aurothioglucose could not be used to predict decreased enzyme activities in vivo. Although decreased activities of two major enzymes that utilize GSH, GSH peroxidase and γ-glutamyl transpeptidase, coincided with elevated GSH in kidneys of aurothioglucose-treated rats, a direct cause and effect relationship remains speculative.

Effect of thyroid hormone on peroxisomal flavin enzymes in rat liver and kidney.

The effect of thyroid hormone on peroxisomal enzyme activity was studied in thyroidectomized- and T4-administered-thyroidectomized rats. In liver, the activities of isozyme A of L-alpha-hydroxyacid oxidase, D-amino acid oxidase, urate oxidase and catalase were decreased by thyroidectomy, and the diminished enzyme activities were restored by T4 administration to rats. These modifications induced by thyroidectomy or by T4 administration, however, were prominent only in immature animals (20-day-old rats). Although the changes in-alpha-hydroxyacid oxidase and D-amino acid oxidase activities, induced by thyroidectomy or by T4 administration, were also observed in 40-day-old rats, those in urate oxidase and catalase activities were not significant in 40-day-old rats. Acyl CoA oxidase activity was not affected by thyroidectomy or by T4 administration in either 20- or 40-day-old rats. In the kidney, isozyme B of L-alpha-hydroxyacid oxidase activity was reduced by thyroidectomy and the diminished enzyme activity was restored by T4 administration in both 20- and 40-day-old rats. D-Amino acid oxidase and catalase activities in kidney, however, were not significantly modified by thyroidectomy or by T4 administration in either 20- or 40-day-old rats. The results suggest that thyroid hormone can modify the peroxisomal enzyme activity, which is prominent in immature animals.

Purification and state of activation of rat kidney phenylalanine hydroxylase.

Phenylalanine hydroxylase, the enzyme that catalyzes the irreversible hydroxylation of phenylalanine to tyrosine, was purified from rat kidney with the use of phenyl-Sepharose, DEAE-Sephacel, and gel permeation high pressure liquid chromatography. Our most highly purified fractions had a specific activity in the presence of 6-methyltetrahydropterin, of 1.5 mumol of tyrosine formed/min/mg of protein, which is higher than has been reported hitherto. For the rat kidney enzyme, the ratio of specific activity in the presence of 6-methyltetrahydropterin to the specific activity in the presence of tetrahydrobiopterin (BH4) is 5. By contrast, this ratio for the unactivated rat liver hydroxylase is 80. These results indicate that the kidney enzyme is in a highly activated state. The rat kidney hydroxylase could not be further activated by any of the methods that stimulate the BH4-dependent activity of the rat liver enzyme. In addition, the kidney enzyme binds to phenyl-Sepharose without prior activation with phenylalanine. The phenylalanine saturation pattern with BH4 as a cofactor is hyperbolic with substrate inhibition at greater than 0.5 mM phenylalanine, a pattern that is characteristic of the activated liver hydroxylase. The molecular weight of the rat kidney enzyme as determined by gel permeation chromatography is 110,000, suggesting that the enzyme might be an activated dimer. We conclude, therefore, that phenylalanine hydroxylases from rat kidney and liver are in different states of activation and may be regulated in different ways.

Effect of mandur bhasma on lipolytic activities of liver, kidney and adipose tissue of albino rat during CCl4 induced hepatic injury

‘Mandur bhasma’, an ayurvedic preparation of iron is used in traditional medicine against hepatitis. In the present study the hepatoprotective property of this drug was tested in albino rats during CC14 induced hepatic injury. The effect of mandur bhasma on the activities of the lipolytic enzymes of rat liver, kidney and adipose tissue were studied during hepatitis induced by CCl4. The activities of acid lipase, alkaline lipase, lipoprotein lipase and hormone sensitive lipase exhibited significant alterations during CCl4 induced hepatic injury, indicating a role for these enzymes in the mobilization of fat from adipose tissue and accumulation of fat in liver and kidney. Simultaneous treatment with mandur bhasma prevented the paraffin mediated and CC14 mediated changes in the enzyme activities. These results suggest the hepatoprotective role of mandur bhasma during CCl4 induced hepatic injury.

The compartmentation of glycolytic and gluconeogenic enzymes in rat kidney and liver and its significance to renal and hepatic metabolism

An indirect immunoperoxidase procedure has been used to demonstrate sites of glycolysis and gluconeogenesis in normal rat kidney and liver. In kidney, the gluconeogenic enzyme fructose 1,6-biphosphatase was restricted to the proximal tubular epithelium, while the glycolytic enzyme hexokinase predominated in more distal segments. Intense staining for the biphosphatase in proximal convoluted tubular brush borders suggests that reabsorbed substrates may be used directly at this site in renal gluconeogenesis. In view of the high phosphofructokinase and pyruvate kinase activities present in collecting ducts, their relatively low hexokinase activities and their relatively pale immunostaining for hexokinase indicate that glycolytic substrates which feed into the pathway subsequent to the initial phosphorylation step, rather than glucose, may be the major energy source for the rat renal papilla. Immunostaining in the liver was consistent with the metabolic zonation of liver parenchyma, in that glucokinase occurred mainly in perivenous regions and fructose 1,6-bisphosphatase in periportal areas. The presence of such metabolic zonation is difficult to reconcile with the widely held view that the majority of hepatic glycogen is derived directly from glucose. A model for hepatic glycogen synthesis is proposed which links the concept of parenchymal zonal heterogeneity with recent biochemical evidence concerning the 'glucose paradox' and with microscopical studies on the dynamics of glycogen deposition after refeeding.

Identification of gamma‐glutamyltransferase in rat liver plasma membranes after two‐dimensional electrophoresis

AbstractTwo‐dimensional electrophoresis and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, followed by silver staining or specific immunoblotting analysis, was applied to the characterization of gamma‐glutamyltransferase (GGT, EC 2.3.2.2.) in rat tissues. We confirmed that purified kidney GGT is a dimer composed of two non‐identical subunits with molecular masses of about 50 and 30 kDa. Both the light and the heavy subunits were separated into 6 and 2 protein bands, respectively. Antibody fractions against the 30 and 50 kDa subunits, purified by immunoaffinity from a whole antiserum directed to the rat kidney enzyme, recognized their corresponding subunits and did not cross‐react with each other. Studies of the reactivity of these antibodies towards GGTs from kidney and liver homogenates revealed an intraspecies dissimilarity in the molecular architecture of the kidney and liver GGTs, especially concerning the 30 kDa subunit. Following phenobarbital treatment of animals we observed an increase in immunoreactive GGT, accompanied by the appearance of additional polypeptides with more basic isoelectric points and higher molecular mass.