General Properties and Substrate Specificity of an Intracellular Neutral Protease From Streptococcus Diacetilactis

An intracellular endopeptidase is isolated from S. diacetilactis.Its molecular weight, deter- mined by gel filtration on Sephadex G-ioo, is 49 500 daltons.Using oxidized insulin as substrate, maximal activity is recorded at 45 0 C with a pH of about !.o ; apparent energy of activation is 1 8 ooo cal/mole.The endopeptidase is most stable at temperatures under 37 °C.It is totally inacti- vated by i mM of EDTA then reactivated by metal ions (Mn ++ or Co ++ ).p-CMB induces a slight reduction in enzyme activity ; Dip-F is not an inhibitor.This protease rapidly attacks oxidized insulin, glucagon or IX 81 -casein but does not appear to hydrolyze proteins (insulin, ribonuclease, p-lactoglobulin, azocasein), the usual esters, mono- or disubstituted dipeptides or substituted amino acids.Endopeptidase specificity is determined by analyzing the peptides in digests of carboxymethylated fi-chain insulin.All the bonds readily cleaving are those involving the a-amino group of hydrophobic residues, i.e.X-Leu or X-Phe.This intracellular enzyme is therefore a a neutral microbial metalloenzyme n (EC 3 .4 -24 -4 ).