Germinating barley grains contain at least eight different peptidases: three carboxypeptidase (pH optima 4.8, 5.2, and 5.7), three aminopeptidases which act on aminoacyl-β-naphthylamides (pH opitima in the hydrolysis of di- and tripeptides at pH 5.8-6.5), and two peptidases which hydrolyse Ala-Gly and Leu-Tyr optimally at pH 7.8 and 8.6 respectively. We have determined the activities of these enzymes in the different tissues of non-germinated grains and followed the changes in the activities during a 5-day germination at 16°C.The aleurone layers contain high activities of all three groups of peptidases; there are no changes in the activities of the five aminopeptidases on germination, while the carboxypeptidases exhibit a small increase of activity. The starchy endosperms contain high carboxypeptidase activities, which increase during germination, but are totally devoid of the five aminopeptidases.All the peptidases exhibit high activities in the scutella; the carboxypeptidases and the enzymes acting on Ala-Gly and Leu-Tyr increase in activity during germination, while the "naphthylamidase" activities remain constant.The three peptidase groups occur in the seedling as well, but compared to the other tissues the carboxypeptidase activities are very small and the "naphthylamidase" activities are very high. The last-named enzymes seem to be characteristic for growing tissues.The starchy endosperm contains about two thirds of the total reserve proteins of the grain. Its internal pH during germination is 5.0-5.2, a value at which all the carboxypeptidases are highly active. As these enzymes are present in high concentrations in this tissue, it is probable that they have a central role in the mobilization of the reserve proteins during germination. The high peptidase activities of the scutellum, on the other hand, suggest that some of the hydrolysis products are absorbed as peptides and these are further hydrolysed to amino acids in this tissue.
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 ...
In germinating grains of barley, Hordeum vulgare L. cv. Himalaya, free proline accumulated in the starchy endosperm during the period of rapid mobilization of reserve proteins. When starchy endosperms were separated from germinating grains and homogenized in a dilute buffer of pH 5 (the pH of the starchy endosperm), the liberation of proline continued in these suspensions. The process was completely inhibited by diisopropylfluorophosphate, indicating that it was totally dependent on serine carboxy-peptidases. The carboxypeptidases present in the starchy endosperms of germinating grains were fractionated by chromatography on DEAE-cellulose. Four peaks were obtained, all with different activity spectra on the seven carbobenzoxydipeptides (Z-dipeptides) tested. Two of the peaks corresponded to previously known barley carboxypeptidases; these as well as a third peak hydrolyzed substrates of the types Z-X-Y and Z-X-Pro (X and Y denote any amino acid residue except proline). The fourth peak corresponded to a proline carboxypeptidase specific for substrates of the Z-Pro-X type. Apparently, in the hydrolysis of longer proline-containing peptides there must be sequential cooperation between the two carboxypeptidase types. The carboxypeptidases in extracts of starchy endosperms also liberated proline from the peptides Ala-Ala-Ala-Pro and Ala-Ala-Pro while Ala-Pro and Pro-Ala were not attacked. The dipeptides, however, were rapidly hydrolyzed around pH 7 by extracts prepared from the scutella of germinating grains. It is concluded that one part of the proline residues of the reserve proteins is liberated in situ in the starchy endosperm through the combined action of acid proteinases and carboxypeptidases, while another part is taken up in the form of small peptides by the scutellum, where proline is liberated by amino- and/or dipeptidases in some "neutral compartment".
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.