Multiple Forms of Acidic Endopeptidase

Abstract

An endopeptidase preparation from germinated barley Hordeum vulgare L., cv. Trophy, purified by affinity chromatography and density-gradient electrofocusing, consisted of three or four components. The preparation was only partly resolved by electrofocusing, with evidence of three possible components (pl 4.15, 4.28, and 4.37). Gel filtration on Sephadex G-75 yielded an asymmetrical peak, the major part of which corresponded to a molecular weight of 14,100, with evidence of one larger and two smaller components. The activity of the preparation was sulfhydryl-dependent; cysteine was the most effective of several sulfhydryl compounds tested. The preparation was sensitive to 02 in the absence of metal chelating agents and was inhibited by sulfhydryl reagents. It showed very narrow concentration tolerances for both cysteine and a substrate, N,N-dimethylhemoglobin. The Km value on N,N-dimethylhemoglobin at pH 3.8 was 0.064 to 0.067% (w/v) substrate; Vm.x was 0.80 to 0.83 A3m0 per hour. Normal enzyme activity and molecular-size distribution were observed when the endopeptidases were extracted in the inhibited state and subsequently reactivated, thus ruling out the possibility that the enzymes might be autolytic artifacts that arose during extraction and purification. Germinated barley and malt contain several endopeptidases (3, 11, 15), most of which are sulfhydryl-dependent (12). Earlier work of this laboratory showed that two of these enzymes from germinated barley, which are active on hemoglobin as substrate at pH 3.8, can be separated from one another by adsorption on and elution from CM2-cellulose at pH 5.5, followed by gel filtration on Sephadex G-100. The remaining enzymes, those that were not adsorbed on CM-cellulose at pH 5.5, were more acidic in character. They were quite similar to one another in their gel-filtration characteristics (3), therefore they could not be separated. Separation methods with higher I Cooperative investigation, Agricultural Research Service, United States Department of Agriculture and the College of Agricultural and Life Sciences, University of Wisconsin, Madison. The Barley and Malt Laboratory is supported in part by a grant from the Malting Barley Improvement Association. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable. C Abbreviations: CM: carboxymethyl; TNBS: trinitrobenzene sulfonic acid; Z-Glu-Tyr: carbobenzoxy glutamyl tyrosine. resolving power were required to purify these enzymes for more detailed study. An obstacle to the study of most of the cereal proteinases is their relative lability under conditions often required for enzyme purification (3, 12). Techniques that are comparatively rapid or that present conditions favorable for retention of enzyme activity are therefore essential. Two such procedures, affinity chromatography and density-gradient electrofocusing, have been used in the present study of the acidic endopeptidases. The results show that these enzymes comprise a group of similar sulfhydryl-dependent endopeptidases that are unusually small and possess rather low isoelectric points. MATERALMS AND METHODS Materials. Barley (Hordeum vulgare L., cv. Trophy) was germinated and lyophilized as previously described (2). All buffers and reagent solutions were prepared in glass-distilled water. Analytical-grade reagents or the purest grades available from commercial sources were used. Hemoglobin-Sepharose was prepared according to Chua and Bushuk (4), with modifications according to Cuatrecasas (7), from twice-crystallized human hemoglobin (Miles Laboratories) and Sepharose 4B (Pharmacia Fine Chemicals). Hordein was prepared from H. distichum L., cv. Piroline, according to Pollack et al. (22). N ,N-dimethylhemoglobin was prepared according to Lin et al. (16). Preparation of Endopeptidases. Lyophilized germinated barley was finely ground in a Labconco mill. Two hundred g were extracted at ice-bath temperature with 300 ml of 0.1 N acetic acid containing 1 mm cysteine. The pH of the slurry was adjusted to 4.1 to 4.2 by the slow addition of glacial acetic acid with constant stirring. After 30 min, the mixture was centrifuged for 1 hr at 14,000g. Affinity chromatography was performed according to the procedure of Chua and Bushuk (4), except that the attachment of the enzyme was accomplished at pH 5.2 rather than pH 5.5. This change in the conditions provided some extra protection for the endopeptidases under study and ensured exclusion of the more anionic endopeptidase activity known to be present in germinated barley (3). The supernatant from the extraction was divided into two equal portions. Each of these was dialyzed at 2 C against two 1300-ml portions of 50 mm sodium acetate buffer, pH 5.2, containing 1 mm cysteine, for 16 hr on a revolving wheel (1 rpm), which operated intermittently: 1 min on, 6 min off. The retentate (about 250 ml) was then centrifuged for 15 min at 14,000g, decanted, and applied by upward flow to a 2.5X 35-cm column of hemoglobin-Sepharose with a pump at 80 ml/hr. This was followed by buffer of the same composition as that used for dialysis, until the optical absorbance at 280 nm of the column eluate had decreased to a constant value. Absorbance was measured with a Vanguard

7 Figures and Tables

Cite this paper

@inproceedings{BurgerMultipleFO, title={Multiple Forms of Acidic Endopeptidase}, author={Warren C. Burger} }