A purification and some properties of two proteases from papaya latex.

Two proteases, one of which is papaya peptidase A and the other a previously unknown enzyme in papaya latex have been purified to homogeneity in a simple two stage process. Both are markedly less reactive than papain or chymopapain. Each has a molecular weight of 24,000, N-terminal sequences commencing Leu-Pro-Glu, and contains no carbohydrate. Their amino acid compositions differ for several residues. The essential -SH groups of the enzymes examined appear to be 'masked' in the native state.

Purification and preliminary characterization of two asclepains from the latex of Asclepias syriaca L. (milkweed).

Two groups of asclepains have been isolated from Asclepias syriaca L. (milk-weed) latex and a representative of each has been purified. Asclepains A3 and B5 are homogeneous proteins with molecular weights of 23 000 and 21 000, respectively. Both require a reducing and chelating agent for maximum activity and hydrolyze ester, amide and peptide bonds. The optimum pH for hydrolysis of casein is 7.5 to 8.5 for asclepain A3 and 7.0 to 7.5 for asclepain B5. Both enzymes are autolytic when active and are inhibited by p-chloromercuribenzoate, iodoacetic acid and sodium tetrathionate. Asclepains A3 and B5 each contain one titratable SH group per molecule and no bound carbohydrate. Each of the two enzymes has leucine as the N-terminal amino acid. There are notable differences in their amino acid compositions.

N-Terminal homology in three cysteinyl proteases from Papaya latex.

Sequences to residue 17 have been determined for the three Papaya cysteinyl proteases, chymopapain and papaya peptidase A and B. Extensive homologies were found for these three enzymes and with papain and bromelain. These results suggest that the five sulphydryl enzymes discussed derive from a common ancestral gene.

Homologies of the N-terminal sequences of asclepains and papain.

Sequences to residue 21 have been determined for the two asclepains, cysteinyl proteases isolated from milkweed (Asclepias syriaca L.). These were compared with the sequence for papain, and extensive homology was found.

Porcine cholesterol esterase, a multiform enzyme.

Cholesterol esterase (sterol-ester acylhydrolase, EC 3.1.1.13) has been purified from porcine pancreas by two methods, one of which was previously reported by Momsen, W.E. and Brockman, H.L. (Biochim. Biophys. Acta. 486 (1977) 102-113). Multiple forms of the enzyme were demonstrated throughout the course of both purification procedures. These forms hydrolyzed both p-nitrophenyl acetate as well as cholesteryl oleate. Isoelectric focusing was used to select one form of cholesterol esterase having a pI of 4.3 for further study. Using high-pressure liquid chromatography on a TSK Spherogel column this apparently homogeneous preparation of cholesterol esterase was separated into two components having molecular weights equal to 90 000 (peak I) and 45 000 (peak II). The number of each amino acid residue in peak I was double that of the corresponding residue in peak II, suggesting a dimer-monomer relationship. The N-terminal analyses showed that the first five amino acid residues were the same in peak I and peak II. The enzyme is a glycoprotein containing glucosamine, glucose, galactose, mannose and rhamnose; it is inhibited by diisopropyl fluorophosphate.

Multiple forms of the asclepains. Cysteinyl proteases from milkweed.

Two groups of asclepains (EC 3.4.22.7) isolated from the latex of Asclepias syriaca L. (milkweed) were each separated into five homogeneous enzymes. The members of each group are of similar amino acid composition, and leucine is the common N-terminal residue. Michaelis values are reported for each of the component cysteinyl proteases of milkweek latex, and are compared with those of analogous enzymes from other plant sources. The asclepains all catalysed the hydrolysis of insulin B chain to yield similar two-dimensional maps. The peptides produced from one such digestion were characterized and scission points were defined and compared with those for papain.

Cross linking in the radiolysis of some enzymes and related proteins.

In non-covalently bound complexes of serveral serine proteases and of ribounclease with DNA the enzymes were protected against the effects of ionizing radiation. No scavenging by the nucleic acids was observed. Similarly, complexing trypsin with silica protected the enzyme from radiolytic destruction. Irradiation of solutions of serine proteases required about twice the D37 dose to produce about 10% polymerization: significantly lower relative doses were effective in causing polymerization in both lima bean protease inhibitor and in the octapeptidal hormone oxytocin. Several sulfhydryl enzymes which have been examined were very efficiently inactivated by ionizing radiation. There was, at the same time, apparent formation of novel intra-molecular -S-S- bonds.