Isolation and Partial Characterization of a Carboxypeptidase from Barley

Abstract

A carboxypeptidase has been purified from germinated barley. The preparations are homogeneous in ultracentrifugal analysis. I n disc electrophoresis traces of impurities can be detected a t high concentration. The purified enzyme liberates carboxyl-terminal amino acid residues from a wide range of carbobenzoxy (Z)-dipeptides. Peptides containing proline are not attacked. It does not possess any endopeptidase, aminopeptidase, or dipeptidase activity. The enzyme has a pH optimum a t pH 5.2, is inhibited by di-isopropylphosphofluoridate and p-chloromercuribenzoate, and has a molecular weight of about 90,000.I n connection with a search for specific synthetic substrates for barley endopeptidases, we observed that freeze-dried enzyme preparations from germinated barley hydrolysed a substrate for collagenase, Z-Gly-Pro-Gly-Gly-Pro-Ala [I], with rapid liberation of the carboxyl-terminal alanine and slower release of the adjacent dipeptide, glycyl-proline [2]. Appaparently, the enzyme preparation contained a carboxypeptidase-like enzyme and an endopeptidase with collagenase-like or very broad specificity.The presence of carboxypeptidase-like activity was confirmed with a series of carbobenzoxy-dipeptides. Several of these, including Z-Leu-Gly, Z-Glu-Tyr, and Z-Pro-Trp, were hydrolysed by the enzyme preparations, Z-Phe-Ala being the compound most rapidly broken down.As the activities were very high and no information on seed carboxypeptidases is available, an endeavour was made to purify the enzyme.We have now isolated the Z-Phe-Ah-hydrolysing enzyme in apparently pure form. The preparation hydrolyses most of the Z-dipeptides tested, and does not possess any endopeptidase, aminopeptidase, or dipeptidase activity. However, it is totally inactive against the original sub strate, Z -Gly -Pro-Gl y-Gl yPro-Ala, and hydrolyses some of the Z-dipeptides with a lower relative rate than the crude enzyme preparations. Apparently barley contains two or possibly several carboxypeptidase-like enzymes, and we have isolated one of them. EXPERIMENTAL PROCEDURES MaterialsPirkka barley (a Finnish 6-row variety) and Pirkka "high enzyme" malt were obtained from Lahden Polttimo Oy (Lahti, Finland). Freeze-dried green malt was prepared from Pirklca barley, as has been described before [3].Reagents and column materials were purchased from the following sources : peptides, Yeda Research and Development Company Ltd., Sigma Chemical Company, Fluka AG, and Hoffman-LaRoche & Co. ; di-isopropylphosphofluoridate, Sigma; DEAE-cellulose (standard, 0.93 mequiv./g), Carl Schleicher & Schull Co. ; CM-cellulose (0.7 mequiv./g), Macherey, Nagel & Go.; Sephadex G-100 and G-200 (for gel filtration), Pharmacia. Other reagents were of reagent grade, except for ammonium sulphate and acetone which were purum grade. Carboxypeptidase AssayZ-Phe-Ala was used as substrate and the alanine liberated was measured by the ninhydrin reaction as follows.1 ml of substrate solution, 2 mM Z-Phe-Ala in 50 mM sodium acetate buffer, pH 5.2, containing 0.5mM EDTA, was added to 0.1 ...

Isolation of a carboxypeptidase from malted barley by affinity chromatography

“…conditions identical to those employed by RAY (38) and VISURE et al (43). From a Lineweaver-Burk plot, which did not deviate from linearity, a K m of 1.9 mM and Vma x of 1400 lamoles/min/mg was obtained.…”

Isolation of carboxypeptidase II from malted barley by affinity chromatography

“…This suggested the existence of five different enzymes which were termed carboxypeptidases I to V. Carboxypeptidase I was purified by VISURI et al (31) and carboxypeptidase II has been partially purified by YABUUCHI et al (34). From the ionic strengths at which the enzymes elute from a DEAE Sepharose column (see section 2.2.3) it is suggested that the enzyme previously isolated by our affinity chromatographic procedure (7) corresponds to malt carboxypeptidase I in MIKOLA'S nomenclature whereas the enzyme described in the present paper corresponds to malt carboxypeptidase II.…”

“…the conditions previously used to characterize purified or partially purified malt carboxypeptidases (28,31,34). From a linear LineweaverBurk plot a Km of 0.53 mM and Vm,x of 132 ttmoles 9 min"mg ~ was obtained.…”

“…Several reports on the purification of carboxypeptidases from malt have appeared (3,28,31,34) but only after the recent introduction of affinity chromatography has it been possible to obtain a homogeneous preparation of one of these enzymes in amounts that allowed a more detailed characterization (7).…”

Purification and Properties of Malt Carboxypeptidases Attacking Hordein

“…These enzymes are also reported to be affected by di-isopropyl fluorophosphate (DIFP). [13][14][15][16][17] In view of the extreme toxicity, and consequent unavailability of this reagent, phenyl methyl sulphonyl fluoride (PMSF) which also inhibits active serinc residues, was used as a substitute. At a final concentration of 2mM, this reagent caused 34% inhibition of carboxypeptidase activity with N-CBZ-Phe-Ala.…”