A proteinase from the sarcocarp of melon (Cucumis Melo L. var. Prince) was purified by a three-step procedure involving batch-wise treatment with CM-cellulose fibers, column chromatography on CM-cellulose powder and gel filtration on Sephadex G-75. The final enzyme preparation was homogeneous on acrylamide gel electrophoresis. Its molecular weight was estimated by two different methods to be about 50,000. Anlayses indicated tha presence of 475 amino acid residues and at least 7 moles of hexose. The maximum activity was found in the alkaline pH region against casein as a substrate. The optimum temperature against casein was 70 degrees at pH 7.1. The enzyme was strongly inhibited by diisopropyl fluorophosphate, partly inhibited by HgCl2 and not inhibited by EDTA, p-chloromercuribenzoic acid, N-tosyl-L-lysine chloromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone, and soybean trypsin inhibitor. The reduced and carboxymethylated insulin B-chain was cleaved at the peptide bonds of Asn3-Gln4, Cm-Cys7-Gly8, Glu13-Ala14, Leu15-Tyr16, Cm-Cys19-Gly20, Phe25-Tyr26, Pro28-Lys29, and Lys29-Ala30 by the enzyme.

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Seven proteinases were isolated from the fruit of snake-gourd, Trichosanthes cucumeroides Maxim. Their isozymes are all serine proteinases, and homologous in their respective molecular weights, amino acid compositions, and enzymatic properties. Their molecular weight was estimated to be about 50,000. Using casein as a substrate, the maximum activity was found in the alkaline pH region. The optimum temperature using casein was 70 degrees C at pH 7.3. The enzymes were strongly inhibited by diisopropyl fluorophosphate and not inhibited by inhibitors of sulfhydryl or metalloproteases. The reduced and S-carboxymethylated insulin B-chain was used as a substrate in an investigation of the specificity. The enzyme was found to have a wide specificity for this substrate but preferentially hydrolyzed the peptide bonds involving the carboxyl groups of charged amino acid such as S-cm-cysteine, glutamic acid, histidine, arginine, and lysine. Experimental evidence indicated that the snake-gourd proteinases are similar in their properties to cucumisin, which is isolated from the sarcocarp of melon fruit.

Cucumisin is a diisopropyl fluorophosphate-sensitive enzyme. The inactivation by DFP is accompanied by the formation of 1 mol of labeled serine residue per mol of enzyme. From the soluble portion of the chymotryptic digest of the diisopropyl phosphoryl derivative of cucumisin, two peptides containing phosphorus were isolated; their amino acid sequences were determined to be Gly-Thr-Ser(P)-Met and Asn-Ile-Ile-Ser-Gly-Thr-Ser(P)-Met, respectively. The four residues Gly-Thr-Ser-Met in the above amino acid sequence are identical with those of subtilisin.