Molecular Forms of Rat‐Liver Arginase. Isolation and Characterization

Abstract

Debate continues over the physical characteristics and even the existence of arginase isoenzymes. This paper gives additional support for such multiplicity and reports differences in physical characteristics among the various forms. After 2500–5000‐fold purification of rat liver arginase, three molecular forms were separated on carboxymethyl‐cellulose columns and were purified 2500–5000‐fold, 800–1000‐fold and 600–1000‐fold, respectively. The molecular forms have also been identified by chromatography in the supernatant of tissue extracts. The isolation of these molecular forms by affinity chromatography, using Sepharose‐lysine as a competitive inhibitor of arginase, shows only one main form, however. Kinetic studies were done for two of the molecular forms isolated, specifically the activation energy (Ea) the energy of denaturatization (Ed), Km, pH and the effect of divalent cations were determined. Significant differences were found for the Ea, between the two molecular forms. The isolated isoenzymes are cationic at pH 5.5 and pH 8.8. However, they show different mobilities in electrophoresis. The molecular weight determination by gel filtration yields a value of 110000 to 115000 for both forms. The use of thin‐layer immunochromatography plates, a combination of molecular weight and immunodiffusion technique, gave only one peak with the same molecular weight as that determined by gel filtration. The immunological studies showed that the isoenzymes have similar antigenic determinants.

Evaluation of optimal conditions for arginase activity in streptozotocin induced diabetic rats

“…The calculated K m value in both control and diabetic groups was 3.16 ± 0.18 mM, similar to the value reported (3 mM) for rat liver enzyme (Tarrab et al, 1974). Jenkinson and Grigor (1994) reported that rat liver and kidney arginases gave the K m values for the substrate, L-arginine, 14.2 and 14.4 mM, respectively.…”

“…The optimum pH for the liver and kidney arginases in both control and diabetic groups is similar to that reported for other mammalian tissues (Tarrab et al, 1974;Konarska and Tomaszewski, 1975;Van Elsen and Leroy, 1975;Spector et al, 1982;Jenkinson and Grigor, 1994). However, the pH profile of arginase in kidney of diabetic rats was different from that of control rats.…”

“…Mn 2+ ions have been found to activate and/or stabilize arginases from different tissues (Kaysen and Strecker, 1973;Tarrab et al, 1974;Konarska and Tomaszewski, 1975;Van Elsen and Leroy, 1975;Spector et al, 1982;Scolnick et al, 1997). Liver and kidney arginases in both control and diabetic groups are also Mn 2+ dependent ( Figure 5).…”

Immunologic Studies of Arginase in Tissues of Normal Human Adult and Arginase-Deficient Patients

“…Rabbit antibody raised against purified rat liver arginase was received as a gift from Dr. Raphael Palacios, Institute of Biomedical Investigation at the Autonomous University of Mexico, Mexlco City, Mexico (15). This antibody precipitated both rat and human liver arginase activity completely, the latter with lower affinity.…”

Sequence heterogeneity of human liver arginase cDNAs and restriction fragment length polymorphism of the gene locus

“…Arginase from rat (Schimke, 1962;Hirsch-Kolb and Greenberg, 1968;Tarrab et al, 1974), rabbit (Vielle-Breitburd and Orth, 1972) and human (Beruter et al, 1978;Brusdeilins et al, 1985) livers has been purified and was found to consist of three identical subunits (Penninckx et al, 1974) of about 35,000 daltons. Arginase isozyme(s) that differs from the liver enzyme in catalytic, molecular and immunological properties, is present in the kidney, small intestine and lactating mammary gland (Glass and Knox, 1973;Herzfeld and Raper, 1976;Skrzypek-Osiecka et aL, 1983;Spector et al, 1983).…”