Anionic Proteinase from Actinidia chinensis. Preparation and Properties of the Crystalline Enzyme

Abstract

1. A method is described for the preparation from Actinidia chinensis (Chinese gooseberry) fruit of a crystalline proteinase which catalyzes the hydrolysis of N2-benzoyl-L-arginine ethyl ester, N2-p-toluene-sulphonyl-L-glutamine p-nitrophenyl ester (used in the routine assay) and a t least 15 o f the peptide bonds of gelatin. The synthesis of the tosyl-glutamine p-nitrophenyl ester is described. The enzyme resembles papain in its action against benzoyl-L-arginine ethyl ester, having a broad pH-optimum from pH 5-7, with K,(app) of 89 mM and keat of about 2.6 sec-l at pH 5.6, 25" in 0.3 M KCI.2. The enzymic activity elutes from Sephadex G-50 as a protein of molecular weight 12800 f 700, and is partially resolved from inactive protein of apparent molecular weight 15400 f 800. The preparation has minimum solubility near pH 3.1 and migrates a t both pH 8.3 and 5.0 on polyacrylamide gel electrophoresis as two closely-spaced anionic bands. These two electrophoretic components are partially resolved by chromatography on DEAE-cellulose and both are enzymically active against tosyl-L-glutamine p-nitrophenyl ester.3. The inhibitions of enzymic activity by 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 4,4'-dithiodipyridine, both reversible by 1,4-dithiothreitol, show that enzymic activity is dependent on an intact sulphhydryl group assaying about 0.23 moles per 12800 g protein. The enzyme is rapidly inactivated by iodoacetate and thereafter gives a low sulphhydryl assay with dithiodipyridine. Conversely, pretreatment with DTNB gives substantial protection against iodoacetate.The proteinase activity in extracts of New Zealand commercial strains of the ripe fruit of the Chinese Gooseberry, Actinidia chinensis has been characterized by Arcus [1] by its activity against gelatin (optimum p H 4 ) and haemoglobin, and was shown to be enhanced by cysteine, Na,S, KCN and EDTA. It is of interest to isolate the enzyme(s) responsible for this activity because of possible structural homologies with the sulphhydryl plant proteinases papain, ficin, bromelin, chymopapain B and pinguanain [2,3], especially now that the tertiary structure of crystalline papain has been elucidated [4].This report describes the preparation of a crystalline anionic proteinase from A. chinensis, some physical and enzymic properties, and further evidence for the requirement of a sulphhydryl group for activity.Unusual Abbreviations. DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid); CNps-proteinase, 3-carboxy-4-nitrophenylsulphenyl-proteinase; BzArgOEt, N2-benzoyl-L-arginine ethyl ester hydrochloride ; TosGlnONp, N2-p-toluenesulphonyl-L-glutamine p-nitrophenyl ester; Pr,P-trypsin, diisopropylphosphoryl-trypsin ; V , and V,, void volume and elution volume.

The interplay of electrostatic fields and binding interactions determining catalytic-site reactivity in actinidin. A possible origin of differences in the behaviour of actinidin and papain

“…The preponderance of anionic side chains over cationic side chains in actinidin provides for its low isoelectric point (3.1) (McDowall, 1970), which contrasts with the much higher value (8.75) for papain. The amino acid residues of both actinidin and papain that contain carboxylic side chains (aspartic acid and glutamic acid) are shown together with the corresponding sequence-aligned residue in the other enzyme in Table 3.…”

Preparation and Structural Analysis of Actinidain-processed Atelocollagen of Yellowfin Tuna (Thunnus albacares)

“…The optimum pH range of actinidain against S-3-trimethylaminopropyl-lysozyme as a substrate is from pH 4 to 8. 23,29) Therefore, the enzyme must be fully active at both pH 4.5 and 6.5. As the self-assembly of atelocollagen increases in the neutral pH range, it might become resistant to hydrolysis by actinidain at pH 6.5.…”

“…[22][23][24][25][26][27] There have been few reports on the collagenolytic activity of actinidain. 28,29) In the present study, therefore, we try to clarify whether actinidain cleaves the inside or outside of the triple helical domain of fish atelocollagen.…”

Actinidain-hydrolyzed Type I Collagen Reveals a Crucial Amino Acid Sequence in Fibril Formation

“…Actinidain is a cysteine protease found in kiwi fruit (Actinidia deliciosa) and is a member of the papain superfamily with a broad specificity toward a variety of substrates (27)(28)(29). We previously found that actinidain-hydrolyzed collagen (AHCol) of tuna retained its triple-helical structure at pH 4.0 and showed the same thermal stability as that of ASCol, as judged by circular dichroism (CD) spectroscopy.…”