Norman P. Curthoys

Specificity of a particulate rat renal peptidase and its localization along with other enzymes of mercapturic acid synthesis

Abstract Renal processing of S -derivatized glutathiones to mercapturic acids requires the participation of three enzymatic activities: γ-glutamyl hydrolase or transpeptidase, a peptidase which is capable of hydrolyzing S -derivatized cysteinylglycine, and an N -acetyltransferase. A particulate peptidase, which was assayed with S -benzylcysteine- p -nitroanilide, was found to be localized along with γ-glutamyltranspeptidase and N -acetyltransferase in the outer stripe region of the renal medulla. This localization suggests that these three activities may be contained primarily in the proximal straight tubules. Results of differential and isopycnic centrifugation indicate that the particulate peptidase is contained along with γ-glutamyltranspeptidase in the brush border membranes while the N -acetyltransferase is probably associated with the endoplasmic reticulum. The partially purified peptidase (200-fold) exhibits a broad substrate specificity. It has greater activity with reduced than oxidized cysteinylglycine, but S -derivatized substrates are hydrolyzed even faster. Comparison of its activity with various substrates indicates that it prefers peptides with a hydrophobic N -terminal amino acid and that it may require a free amino group. Heat-inactivation studies suggest that all of these activities are attributable to a single enzyme. These results suggest that this peptidase may participate along with γ-glutamyltranspeptidase and an N -acetyltransferase in the conversion of glutathione conjugates to mercapturic acids.

The interorgan metabolism of glutathione

Abstract 1. 1. Glutathione is the primary low molecular weight cellular thiol and a major reserve of cysteine. Glutathione is synthesized in the cytoplasm, where it participates in a variety of reactions as an electron donor or a nucleophilic cofactor. 2. 2. However, it now appears that the initial step in glutathione turnover is its release from the cell. The released glutathione is transported by the blood plasma to the kidney where it is efficiently extracted and degraded to its constituent amino acids. 3. 3. The initial reaction in the renal degradation of glutathione is catalyzed by y-glutamyltranspeptidase. which is localized on the external surface of the brush border membrane. 4. 4. Within the lumen of the renal proximal tubule, the γ-glutamyltranspeptidase apparently catalyzes a hydrolytic rather than a transpeptidation reaction. 5. 5. The resulting eysteinylglycine is hydrolyzed by two extracellular brush border membrane enzymes. aminopeptidase M and a recently identified peptidase. 6. 6. Reabsorption of the amino acids and redistribution of cysteine for protein synthesis or glutathione resynthesis completes the interorgan metabolism of glutathione.

Regulation of renal ammoniagenesis. Purification and characterization of phosphate-dependent glutaminase from rat kidney

Abstract Phosphate-dependent glutaminase can be solubilized from rat kidney mitochondria by treatment with high concentrations of nonionic detergents or by lyophilization in the presence of a borate buffer. Borate promotes a reversible polymerization of this enzyme into a high molecular weight aggregate. Removal of borate to produce the low molecular weight Tris form of glutaminase is associated with a reversible threefold decrease in specific activity. This process makes possible the rapid purification (1000-fold) of this glutaminase by successive gel filtration in each of its molecular weight forms. The purified glutaminase is stable at 3 °C in borate, phosphate buffer for at least 6 months. Activation of this enzyme is not specific and can be achieved by a large number of phosphate-containing compounds including pyrophosphate, nucleotides and sugar phosphates. Nucleotides produced a significantly greater percentage activation of the mitochondrial and borate forms of glutaminase than of the Tris form. In addition, only the former two forms are activated by a large number of carboxylic acids. The specific activities of the borate and Tris forms of glutaminase are also increased by the presence of Triton X-100.

Phosphate-dependent glutaminase from rat kidney: Cause of increased activity in response to acidosis and identity with glutaminase from other tissues☆

Abstract Immune serum was prepared against phosphate-dependent glutaminase purified from rat kidney and was used to investigate the cause of increased renal glutaminase activity in acidotic rats. Crude kidney homogenates from acidotic rats exhibited a fourfold greater specific activity for phosphate-dependent glutaminase. The glutaminase was solubilized initially by lyophilization of borate treated mitochondria with a 40–60% recovery and with maintenance of threefold difference in specific activity. Both preparations showed the same equivalence point in a quantitative precipitin experiment. To confirm these results, phosphate-dependent glutaminase was also solubilized by treatment of mitochondria isolated from normal and acidotic rat kidney cortex with 1% Triton X-100. The two preparations exhibited a fivefold difference in specific activity and again showed the same equivalence point in a quantitative precipitin experiment. These results indicate that the cause of increased phosphate-dependent glutaminase activity during acidosis is due to the presence of an increased amount of this enzyme. The antiserum prepared against the kidney phosphate-dependent glutaminase did not crossreact with glutaminase solubilized from rat liver mitochondria. But, rat brain mitochondria do contain a phosphate-dependent glutaminase that is immunologically identical to the enzyme from rat kidney.

Immunocytochemical localization of glutaminase-like and aspartate aminotransferase-like immunoreactivities in the rat and guinea pig hippocampus

There is considerable evidence that pathways of the hippocampus use an excitatory amino acid as transmitter. We have attempted to immunocytochemically identify excitatory amino acid neurons in the hippocampus of the rat and guinea pig using antiserum to glutaminase and antiserum to aspartate aminotransferase, which have been proposed as markers for aspartergic/glutamergic neurons. Glutaminase-like immunoreactivity was seen in granule cells in the dentate gyrus and fibers and puncta associated with the mossy fiber pathway in the hilus and stratum lucidum of the hippocampus. At the ultrastructural level, glutaminase-like immunoreactivity was observed in mossy fiber terminals in the stratum lucidum. Glutaminase-like immunoreactivity was also seen in pyramidal cells in regio inferior and regio superior and in cells in layer two of the entorhinal cortex. Schaffer collateral terminals, commissural fiber terminals and perforant pathway terminals were not seen at the light microscopic level. Glutaminase-like immunoreactivity is thus found in the cell bodies of proposed excitatory amino acid neurons of hippocampal pathways, but does not appear to label all terminals. Aspartate aminotransferase-like immunoreactivity was not seen in any cells, fibers or terminals in the rat or guinea pig hippocampus.

Immunocytochemical localization of glutaminase-like immunoreactivity in the auditory nerve.

The immunocytochemical localization of glutaminase, which we have proposed as a marker for excitatory amino acid neurotransmitters was determined in the guinea pig auditory nerve. Glutaminase-like immunoreactivity was seen in auditory nerve terminals in the cochlear nucleus and in the cell bodies of the auditory nerve in the cochlea. This staining was seen in type I and not type II spiral ganglion cells. Glutaminase-like immunoreactivity was also observed in granule cells in the cochlear nucleus.

Localization of elevated glutaminase immunoreactivity in small DRG neurons

Glutamate has long been considered to be a neurotransmitter candidate in vertebrate spinal sensory nerve cells. We report here the first immunohistochemical evidence in support of this hypothesis. We find that up to 30% of the moderately small dorsal root ganglion neurons in the rat contain elevated levels of glutaminase immunoreactivity. This enzyme, which mediates the synthesis of glutamate from glutamine, is not found at these high levels in large diameter neurons of the same ganglia. In contrast, another enzyme associated with glutamate metabolism, aspartate aminotransferase, is rather uniformly distributed within neurons of the sensory ganglia. These data define a subpopulation of sensory neurons which appear to contain an elevated capacity to synthesize glutamate through the glutamine cycle and suggest that glutaminase immunoreactivity may be an indicator of glutamatergic function in some nerve cells.

Evidence for the renal paratubular transport of glutathione

The catabolism of glutathione in various nonrenal tissues occurs through an interorgan process [ 1,2]. For example, the turnover of glutathione within rat liver is initiated by the unidirectional release of glutathione to the blood plasma [3]. The released glutathione is carried to the kidney where it is nearly quantitatively extracted and degraded to its constituent amino acids [4,5]. The glutathionemia and the pronounced glutathionuria observed in a patient who lacks detectable -r-glutamyltranspeptidase [6] and in mice treated with inhibitors of y-glutamyltranspeptidase [7,8] indicate that this enzyme catalyzes the initial reaction in glutathione catabolism. The transpeptidase is an amphipathic membrane glycoprotein [9] that is found to the greatest extent in the kidney. Within this tissue, the enzyme is primarily associated with the brush border membrane of the proximal tubule cells [lo], where it is asymmetrically orientated on the lumenal surface [ 111. the glomeruli even when the arterial glutathione concentration is increased to a value 200-fold greater than normal [5]. Therefore, the kidney may contain a mechanism for the transport of glutathione across the basolateral plasma membrane. However, small amounts of y-glutamyltranspeptidase may be associated with the glomeruli [13], with the renal microvasculature [14], or with the basolateral membrane of the proximal tubule [ 151. Thus, the apparent extraction of glutathione by the kidney may be due to its catabolism within the post-glomerular paratubular space. In order to resolve these two possibilities, we have used the procedures in [16] to study the renal paratubular handling of glutathione.

The orientation of phosphate-dependent glutaminase on the inner membrane of rat renal mitochondria

Phosphate-dependent glutaminase is associated with the inner membrane of rat renal mitochondria. The orientation of this enzyme was characterized by comparing its sensitivity in isolated mitochondria and in mitoplasts to two membrane impermeable inhibitors. Mitoplasts were prepared by repeated swelling of mitochondria in a hypotonic phosphate solution. This procedure released greater than 70% of the adenylate kinase from the intermembrane space, but less than 10 and 25% of the marker activities characteristic of the inner membrane and matrix compartments, respectively. The addition of 20 microM p-chloromercuriphenylsulfonate (pCMPS) caused a rapid inactivation of the purified glutaminase. In contrast, the glutaminase contained in isolated mitochondria and mitoplasts was only slightly affected by the addition of up to 2 mM pCMPS. Similarly, the activity in mitochondria and mitoplasts was not inhibited by the addition of an excess of inactivating Fab antibodies. However, a similar extent of inactivation occurred when either membrane fraction was incubated with concentrations of octylglucoside greater than 0.35%. Mitochondria were also treated with increasing concentrations of digitonin. At 0.4 mg digitonin/mg protein, all of the adenylate kinase was released but the glutaminase activity was either slightly inhibited or unaffected by the addition of pCMPS or the Fab antibodies, respectively. These studies establish that the glutaminase is localized on the inner surface of the inner membrane. Therefore, mitochondrial catabolism of glutamine must occur only within the matrix compartment.

Subcellular localization of rat kidney phosphate independent glutaminase

Abstract The phosphate independent glutaminase is contained in the brush border membrane of the rat kidney proximal tubule cells. This glutaminase activity cofractionates with the brush border membrane marker activities, alkaline phosphatase and γ-glutamyltranspeptidase, during differential centrifugation. About 30% of these activities are recovered with the mitochondrial fraction, the remainder is pelleted in the heavy microsomal fraction. The phosphate independent glutaminase in both fractions bands, during isopycnic centrifugation, with a mean density of 1.16–1.17 and is coincident with both brush border membrane marker activities. The isolation of intact, individual kidney cells was accomplished by initial perfusion of the kidneys in situ with a collagenase-papain solution followed by a brief incubation in the same enzyme solution. Incubation of isolated cells with a higher concentration of papain results in selective release of the phosphate independent glutaminase. The fact that this occurs without appreciable release of a cytoplasmic marker activity, lactate dehydrogenase, suggests that the phosphate independent glutaminase may be localized on the external surface of the kidney cells.