L‐Asparaginase activity in Leptosphaeria michotii. Isolation and properties of two forms of the enzyme

Abstract

Jerebzoff -Ouintin, S. and Jerebzoff, S. 1985. L-Asparaginase activity in Leptospkaeria michotii. Isolation and properties of two forms of the enzyme, -Physiol, Plant, 64: 74-80.The properties of L-asparaginase (EC 3.5.1.1) in Leptosphaeria michotii (West) Sacc, which has previously been shown to have an activity rhythm, were analyzed. Two forms of L-asparaginase were isolated from acetic acid and ammonium sulfate fractionations followed by DEAE-Sephacel chromatography. The activity of L-asparaginase changed rhythmically with the same period as that of crude extracts, but the rhythms of the two enzyme forms were out of phase. The two asparaginase forms differed in their isoelectric points and the substrate concentrations for attaining halfmaximal velocity; non-Michaelis-Menten kinetics for hydrolysis of L-asparagine were observed. Analyses of asparaginase form II by polyacrylamide gel eiectrophoresis showed that four proteins, irrespective of the phase of the activity rhythm at which the enzyme was extracted, could be detected: asparaginase oligomer (M, I300O0 to 140000), its dimer, an aggregate (M, 500000 to 600000) having a low asparaginase activity, and a protein (M^ 60000) without asparaginase activity; the same proteins were found in asparaginase form 1. These results indicate that L. michotii asparaginase could be implicated in a protein complex.Additional key words -Asparagine, fungus, rhythmic enzyme activity, oscillating system. 5*. Jerebioff-Quintin and S. Jerebzoff (reprint requests). Lab.

Transaminase activity rhythms in Penicillium claviforme

“…Asparaginase (EC 3.5.1.1) was assayed according to Jerebzoff-Quintin and Jerebzoff (1985), the activity being determined by the amount of [U-I4 C] aspartate formed from [U-14 C] asparagine. The radioactivity was determined in a Packard CSL 460C scintillation spectrometer.…”

Changes in protein kinase and protein phosphatase properties during the cycle of asparaginase activity in Leptosphaeria michotii

“…(1) For control assays, the asparaginase (Lasparagine amido-hydrolase, EC 3,5.1,1) protein complex was isolated and purifted according to Jerebzoff-Quintin and Jerebzoff (1985). A form I, detected in the void voltime of the DEAE-Sephacel column, and a more abundant and more purified form II, eluted from the column with 0.4 M NaCl, were obtained.…”

Cyclic activity of L‐asparaginase through reversible phosphorylation in Leptosphaeria michotii

“…This rhythm has been shown to be regulated at two levels: (a) protein synthesis on 80 S ribosomes; @) the asparagine-pyruvate pathway, 3 enzymes of which, namely asparaginase and aspartate and alanine aminotransferases, expressed activity rhythms having a period identical with that of the sporulation rhythm [ 1,2]. Asparaginase activity rhythm, which played a very important part in this periodic system, continued in crude extracts in spite of a dramatic inhibition of protein synthesis [3]; this rhythm was not suppressed in partially (200-fold) purified extracts mainly constituted of form I and form II asparaginases (130)(131)(132)(133)(134)(135)(136)(137)(138)(139)(140) and their polymers (270 and 500-600 kDa) and a -60-kDa protein [4]. All these facts supposed that posttransductional protein modifications could be implicated in the regulation of the rhythmic asparaginase activity in L. michotii.…”