Substrate inhibition of rat liver and kidney arginase with fluoride

Fluoride is an uncompetitive inhibitor of rat liver arginase. This study has shown that fluoride caused substrate inhibition of rat liver arginase at substrate concentrations above 4 mM. Rat kidney arginase was more sensitive to inhibition by fluoride than liver arginase. For both liver and kidney arginase preincubation with fluoride had no effect on the inhibition. When assayed with various concentrations of L-arginine, rat kidney arginase did not have Michaelis-Menten kinetics. Lineweaver-Burk and Eadie-Hofstee plots were nonlinear. Kidney arginase showed strong substrate activation at concentrations of L-arginine above 4 mM. Within narrow concentrations of L-arginine, the inhibition of kidney arginase by fluoride was uncompetitive. Fluoride caused substrate inhibition of kidney arginase at L-arginine concentrations above 1 mM. The presence of fluoride prevented the substrate activation of rat kidney arginase.     

Chemical modification of rat liver arginase

Chemical modifications were used to search for catalytically important residues of rat liver arginase. The results of carbamoylation, nitration and diazotization suggest that lysyl and tyrosyl residues are not involved in the catalytic function of arginase. The modification of 5--6 tryptophanyl residues by N-bromosuccinimide or 2-hydroxy-5-nitrobenzyl bromide led to about 90% inhibition of the enzyme activity. Photooxidation of 21 histydyl residues also led to considerable inactivation of arginase. The modification of tryptophanyl and histidyl residues did not cause dissociation of the enzyme into subunits.     

Circular dichroic properties of rat liver arginase

• 1.1. Rat liver arginase contains over 50% of the α-helical and about 10% of β-pleated structures.
• 2.2. The manganese ions do not cause the changes in the far ultraviolet CD spectra of the enzyme, whereas they induce the optical activity at 280 nm.
• 3.3. The circular dichroic changes at near ultraviolet region coincide with the activation of arginase.

     

Hybridization of subunits of rat liver arginase A1 and rat kidney arginase A4

Recombination of subunits of rat liver arginase A1 and rat kidney arginase A4 yielded a product which in polyacrylamide gel electrophoresis and DEAE-cellulose chromatography separated into five proteins with arginase activity. Proteins I and V corresponded in polyacrylamide gel-electrophoresis, DEAE-cellulose chromatography and immunological properties to the parental forms A1 and A4, respectively. Formation of five arginase hybrids proved the tetrameric structure of native arginases.     

Growth inhibitory and arginase activities in liver and hepatoma extracts.

The growth-inhibitory activity against H.Ep. No 2 cells observed in phosphate buffer extracts of rat liver appears to be due to the enzyme, arginase. This conclusion is based on the findings that (1) extracts of hepatomas containing lower levels of the enzyme also contain less inhibitory activity; (2) partial purification of the inhibitory factor results in a similar purification of the enzyme; (3) molecular weights of the enzyme and of the inhibitory factor are similar; and (4) various treatments alter the enzyme and inhibitory activities similarly.     

Glutamate-supported calcium movements in rat liver mitochondria effects of anions and pH

Ca²⁺ efflux from rat liver mitochondria in the presence of glutamate is stimulated by a decrease in pH from 7.3 to 6.8 and the rate is dependent on the phosphate concentration. During Ca²⁺ (13 μm) uptake and release at low pH (+ phosphate), swelling is minimal, but a large oxidation of pyridine nucleotides and sustained membrane depolarization occurs. The depolarization (but not Ca²⁺ efflux) is reversed by ruthenium red. An absolute requirement for phosphate to support Ca²⁺ efflux is demonstrated by using acetate or lactate to support Ca²⁺ uptake (efflux is depressed at pH 6.8). Preincubation with mersalyl, to block phosphate movements, with subsequent phosphate addition preceeding Ca²⁺ uptake also inhibits efflux. β-Mercaptoethanol then stimulates efflux concomittent with membrane repolarization. Ca²⁺ efflux is not a simple result of collapse of ΔpH since nigericin inhibits phosphate transport and Ca²⁺ release. Following Ca²⁺ uptake at pH 6.8, respiratory inhibition occurs, but oxygen consumption coupled to ATP synthesis can be stimulated by succinate (+ rotenone). Addition of succinate allows reuptake of Ca²⁺, reduction of pyridine nucleotides, and repolarization of the membrane potential. Respiratory inhibition is also seen with nigericin, but no Ca²⁺ efflux is observed. Coupled respiration with glutamate is seen at pH 6.8 following Ca²⁺ uptake in the presence of lactate with subsequent addition of phosphate to promote Ca²⁺ efflux. We conclude that Ca²⁺ efflux is not a consequence of respiratory inhibition, but is mediated solely by phosphate movements. The inhibitory effect of Mg²⁺ on Ca²⁺ efflux is probably due to Mg²⁺-dependent inhibition of the Ca²⁺ diffusion potential so that the compensatory increase in ΔpH due to membrane depolarization does not occur and phosphate entry is slowed.     

Crystallization and oligomeric structure of rat liver arginase.

Rat liver arginase, a manganese-metalloenzyme, has been crystallized from polyethylene glycol 8000 in N,N-bis(2-hydroxyethyl)glycine (Bicine) buffer at pH 8.5. Crystals form as either cubes or pyramids and belong to space group P3(1) (or P3(2)) with hexagonal unit cell dimensions a = b = 88.9 A, c = 114.8 A, or a = b = 88.5 A, c = 104.5 A; the variation along the c axis does not correlate with the external crystal morphology of cube or pyramid-shaped. X-ray diffraction data are measured to a limiting resolution of 2.4 A. Given the volume constraints of the unit cell it is likely that rat liver arginase is a trimer, with three 35,000 Da monomers in the asymmetric unit. This resolves a persistent ambiguity regarding the oligomeric structure of this enzyme.     

Inhibition of rat liver and kidney arginase by cadmium ion

Cadmium ion activates arginase from many species of organisms but is an inhibitor of arginase from many other species. The purpose of this study was to investigate the inhibition of rat liver and kidney arginase by cadmium ion. Rat kidney arginase was inhibited by much lower concentrations of cadmium ion than rat liver arginase. Cadmium ion was a mixed noncompetitive inhibitor of both rat liver and kidney arginase. Cadmium ion enhanced the substrate activation of rat kidney arginase while still inhibiting the enzyme. Cadmium ion prevented the substrate inhibition of rat kidney arginase by fluoride while still inhibiting the enzyme. Cadmium ion also inhibited rat kidney arginase in the presence of manganese ion.     

Production and characterization of monoclonal antibodies to rat liver arginase

Rat liver arginase was purified and five monoclonal antibodies were produced by fusion of spleen cells from a Balb/c mouse and the myeloma cell line P3-X36-Ag-U1. One, R2D19, of five antibodies belonged to the IgG2a subclass, the other four, R1D81, R1G11, R2E10, and R2G51, were of the IgG1 type. The R1D81 cross-reacted with human liver arginase. This antibody inhibited the arginase activity, competing with arginine. These results suggest that R1D81 binds to the catalytic site of arginase. The R2D19 also inhibited the enzyme activity but acted as a noncompetitive inhibitor. With the use of R1D81 and a polyclonal anti-human liver arginase antibody conjugated with alkaline phosphatase, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of human arginase. Specificity of monoclonal antibodies for rat liver arginase was examined by means of the sandwich ELISA. Eight pairs of monoclonal antibodies could form a sandwich with the arginase. Only the R2E10 could be used for both the first and the second antibody in the sandwich system. In other cases, monoclonal antibodies could not be interchanged between solid and liquid phase.     

Inhibition of rat liver and kidney arginase by cadmium ion

Cadmium ion activates arginase from many species of organisms but is an inhibitor of arginase from many other species. The purpose of this study was to investigate the inhibition of rat liver and kidney arginase by cadmium ion. Rat kidney arginase was inhibited by much lower concentrations of cadmium ion than rat liver arginase. Cadmium ion was a mixed noncompetitive inhibitor of both rat liver and kidney arginase. Cadmium ion enhanced the substrate activation of rat kidney arginase while still inhibiting the enzyme. Cadmium ion prevented the substrate inhibition of rat kidney arginase by fluoride while still inhibiting the enzyme. Cadmium ion also inhibited rat kidney arginase in the presence of manganese ion.     

Interaction of rat liver glucocorticoid receptor with heparin

When rat liver cytosol containing [3H]dexamethasone-glucocorticoid receptor complex is exposed to immobilized heparin (Sepharose-heparin; Seph-hep) the steroid receptor complex binds to the substituted Sepharose avidly [Kd = 3.5 (+/- 1.7) X 10(-10) M], and 80-90% of the receptor present is adsorbed to the solid phase after 40 min at 0 degree C. The binding is enhanced by Mn2+ (10 mM) and Mg2+, whereas Ca2+ and Sr2+ are ineffective. Sodium molybdate (10 mM) does not influence the reaction but enhances receptor stability. Moreover, binding of the receptor to Seph-hep is dependent on the ionic strength of the medium, because binding is totally reversed by 300 mM KCl. The bound [3H]dexamethasone-receptor complex can be recovered from Seph-hep with solutions (4 mg/mL) of heparin (95% release), dextran sulfate (88%), and chondroitin sulfate (63%); total calf liver RNA is less effective (9%), whereas dextran, D-glucosamine, N-acetyl-D-glucosamine, D-glucuronic acid, and sheared calf thymus DNA are totally ineffective (less than 3%). Both "native" and temperature "transformed" forms of the glucocorticoid receptor interact with immobilized heparin. These results strongly suggest that the receptor site that binds heparin is distinct from that binding DNA. An immediate application of this newly found ability of the glucocorticoid receptor to interact with heparin is the use of Seph-hep for affinity chromatography purification of the glucocorticoid receptor. A purification of 10-fold, with a recovery of 55-65%, can be achieved by using either 4 mg/mL heparin or 300 mM KCl to elute [3H]dexamethasone-receptor bound to the resin.     

Interaction of rat liver lysosomal membranes with actin

Membranes were prepared from lysosomes purified 80-fold by centrifugation in a discontinuous metrizamide gradient. When salt- washed membranes were combined with rabbit muscle actin, an increase in viscosity could be measured using a falling ball viscometer. The lysosomal membrane-actin interaction was actin- and membrane- concentration dependent and appeared to be optimal under presumed physiological conditions (2 mM MgCl2, 1 mM MgATP, neutral pH, and free calcium concentration less than 10(-8) M). The actin cross-linking activity of the membrane was optimal at pH 6.4. The interaction was maximal between 10(-7) and 10(-9) M free calcium ions and inhibited by approximately 50% at concentrations of calcium greater than 0.5 x 10(- 7) M. The actin-lysosomal membrane interaction was destroyed if the membranes were pretreated with Pronase, or if the membranes were purified in the absence of protease inhibitors. The interaction was not destroyed if membranes were washed with high salt or extracted with KCl and urea. In addition, a sedimentation assay for the actin-lysosomal membrane interaction was also performed to corroborate the viscometry data. The results suggest the existence of an integral lysosomal membrane actin-binding protein.