Shigeo Tomioka

A catalytic subunit of calpain possesses full proteolytic activity

Previous studies on the refolding of calpain, a heterodimer comprising a catalytic 80 kDa subunit and a regulatory 30 kDa subunit, indicate that both subunits are required for the expression of full protease activity. We reexamined the conditions for refolding of calpain and found that under optimized conditions the renatured 80 kDa subunit has full enzyme activity even in the absence of the 30 kDa subunit. The 30 kDa subunit stabilizes the 80 kDa subunit rather than enhancing its activity. The theory that calpain functions as a dimer requires reexamination.

Autolysis of calpain large subunit inducing irreversible dissociation of stoichiometric heterodimer of calpain

Calpain, a calcium dependent cysteine protease, consists of a catalytic large subunit and a regulatory small subunit. Two models have been proposed to explain calpain activation: an autolysis model and a dissociation model. In the autolysis model, the autolyzed form is the active species, which is sensitized to Ca2+. In the dissociation model, dissociated large subunit is the active species. We have reported that the Ca2+ concentration regulates reversible dissociation of subunits. We found further that in chicken μ/m-calpain autolysis of the large subunit induces irreversible dissociation from the small subunit as well as activation. So we could propose a new mechanism for activation of the calpain by combining our findings. Our model insists that autolyzed large subunit remains dissociated from the small subunit even after the removal of Ca2+ to keep it sensitized to Ca2+. This model could be expanded to other calpains and give a new perspective on calpain activation.

Purification of penicillin-insensitive DD-endopeptidase, a new cell wall peptidoglycan-hydrolyzing enzyme in Escherichia coli, and its inhibition by deoxyribonucleic acids

Abstract Activity of a penicillin-insensitive DD -endopeptidase that splits the D -alanyl-meso-2,6-diaminopimelyl linkage in peptidoglycan was demonstrated in a sonic extract of Escherichia coli . The protein with this activity was partially purified. The activity was inhibited by 3 μg per ml of deoxyribonucleic acid, suggesting that this cell wall hydrolytic enzyme is regulated by deoxyribonucleic acid or its fragments.

Formation of hyper-crosslinked peptidoglycan with multiple crosslinkages by a penicillin-binding protein, 1A, of Escherichia coli

Abstract Hyper-crosslinked peptidoglycan was synthesized in vitro by purified penicillin-binding protein 1A of Escherichia coli . The peptidoglycan formed was crosslinked up to 39%. About half the crosslinks were novel three-handed crossbridges whereas the other half were two-handed crossbridges that are the major constituents of normally crosslinked peptidoglycan of E. coli . The structure of the three-handed crossbridge constructed among three peptide side-chains of - l -alanyl- d -glutamyl-meso-diaminopimelyl- d -alanyl- d -alanine was deduced from several criteria. Probably penicillin-binding protein 1A is responsible for hyper-crosslinking of E. coli peptidoglycan in vivo .

Purification and characterization of the plasmodial phosphatase that hydrolyses the phosphorylated light chain of Physarum myosin II from Physarum polycephalum.

A phosphatase was purified through a combination of ion‐exchange and hydrophobic chromatography followed by native PAGE from Physarum plasmodia. Recently, we demonstrated that this phosphatase isoform has a hydrolytic activity towards the PMLC (phosphorylated light chain of Physarum myosin II) at pH 7.6. The apparent molecular mass of the purified enzyme was estimated at approximately 50 kDa by means of analytical gel filtration. The enzyme was purified 340‐fold to a final phosphatase activity of 400 pkat/mg of protein. Among the phosphorylated compounds tested for hydrolytic activity at pH 7.6, the enzyme showed no activity towards nucleotides. At pH 7.6, hydrolytic activity of the enzyme against PMLC was detected; at pH 5.0, however, no hydrolytic activity towards PMLC was observed. The K m of the enzyme for PMLC was 10 μM, and the V max was 1.17 nkat/mg of protein. Ca2+ (10 μM) inhibited the activity of the enzyme, and Mg2+ (8.5 μM) activated the dephosphorylation of PMLC. Mn2+ (1.6 μM) highly stimulated the enzymes activity. Based on these results, we concluded that the enzyme is likely to be a phosphatase with hydrolytic activity towards PMLC.