Proteinase activity, alkaline

In an earlier report (J>), the decay of healthy yam tubers during storage was shown to be a result of catabolism of its proteins by an active a-glutamyl transpeptidase. There is also some alkaline proteolytic activity in the yam tuber (6), but little information is available on individual enzymes of the purine degradative pathway and on the properties of an alkaline proteinase that may function in yams during storage. This report describes the interrelation of five enzymes of ureide metabolism in fresh and stored yams, the release of ammonia in vitro by three of the enzymes that may provide an environment for alkaline proteinase activity in vivo, and the in vitro properties of an... 

Alkaline Proteinase Activity in Yams. The release of ammonia at several stages during ureide metabolism suggested a potential for alkaline conditions in yam tubers, rather than the usual neutral or acid conditions generally found in seeds and plants. 

Because yams are stored in open systems at ambient temperatures (usually warm), tuber tissue was examined for proteinase activity at 40°C. Some tubers had high apparent polyphenol oxidase activity upon peeling of the tubers (tissue turned deep purple at the peeled surface) so that PYP was added to extracts to combine with polyphenolic compounds and protect the proteinase from reacting with these compounds. Earlier studies had shown some inhibition of alkaline proteinase activity by ferric ion (24) so that EDTA was also added to the extracts to chelate any free iron. Two alkaline pH optima were found, at 9.0 and 10.5. The alkaline proteinases of white potatoes (Solanum tuberosum) have pH optima between 8.6 and 9 (25) and those of Carilla chocola tubers have pH optima between 8.0 and 9.5 (26,27T, suggesting that alkaline... 

Table 4. Substrate Specificity of Yam Tuber Alkaline Proteinase (Activity mg protein hydrolyzed)/10 mg enzyme/2hr).

Activity of plasmin (indigenous milk alkaline proteinase). 

Its volume practically does not change on variation of pH, ionic strength, and in the presence of some organic solvents. It may be sterilized. It cannot be used, of course, in the presence of cellulases. For the immobilization of bacitracin on the cellulose we used processes that give rise to a stable bond of the ligand to the polysaccharide activation of the cellulose with 2, 4, 6-trichlorotriazine or benzoquinone directly or by diazo-tization of a 2-(4-aminophenylsulfonyl) ethyl derivative of cellulose. Bacitracin-celluloses were used for the purification of subtilisin DY and for isolation of alkaline proteinase from the culture medium of Bacillus subtllis. The best results were obtained with a biospecific adsorbent prepared by the attachment of bacitracin to bead cellulose activated by 2,4,6-trichlorotriazine. 

Enzymes of this group, in which activity is confined to the pH range of 7—11, are denoted as alkaline proteinases. Typical representatives... 

Milk contains at least two proteinases, plasmin (alkaline milk proteinase) and cathepsin D (acid milk proteinase) and possibly several others, i.e. two thiol proteinases, thrombin and an aminopeptidase. In terms of activity and technological significance, plasmin is the most important of the indigenous proteinases and has been the subject of most attention. The relevant literature has been reviewed by Grufferty and Fox (1988) and Bastian and Brown (1996). 

It is possible to use ODVs as a source of infectious virions if they are released from the OBs using alkaline solutions. But when OBs are dissolved in alkaline solution before infection of cell lines, there is only a low level of in vitro activity (Vail et al., 1979 Elam et al., 1990). However, this process can be improved by using proteinases. McIntosh et al. (1988) achieved an improvement in the insect cell culture susceptibility to ODVs using a treatment with proteinase K to dissolve Helicoverpa zea NPV (HzSNPV) polyhedra, which could infect homologous cells. Lynn (1994) also achieved the same result when he used a treatment of dissolving Lymantria dispar NPV (LdMNPV) polyhedra with trypsin. 

Melloni et al. (22, 23) obtained three distinct enzymes from rabbit liver lysosomes that catalyze limited proteolysis of rabbit liver fructose-1,6-bisphosphatase, converting the neutral form to a form with an alkaline pH optimum. One of these proteinases (Mr = 70,000) is associated with the lysosomal membrane fraction. Since cathepsin B, H, and L are all present in the soluble fraction of lysosomes 61), this enzyme seems to be a new thiol proteinase of lysosomes. The enzyme is activated by cysteine, but not inhibited by leupeptin. Since it has only... 

Subtilisin (EC 3.4.21.4) an extracellular, single chain, alkaline serine protease from Bacillus subtilis and related species. S. are known from four different species of Bacillus S. Carlsberg (274 amino acid residues, M, 27,277), S. BPN (275 amino acid residues, M, 27,537), S. Novo (identical with S.BPN ) and S. amylosacchariticus (275 amino acid residues, M, 27671). The observed sequence differences between different S. represent conservative substitutions and are limited to the surface amino acids. Like the pancreatic proteinases, S. has catalytic Ser22i, His64 and Asnjj residues, but it is structurally very different from the other serine proteases, e. g. the active center of S. is -Thr-Ser-Met-, whereas that of the pancreatic enzymes is -Asp-Ser-Gly- pancreatic enzymes contain 4- disulfide bridges, whereas S. contains none S. contains 31 % a-helical structure and 3 spatially separated domains, whereas the pancreatic enzymes have 10-20% a-helical structure and a high content of p-structures in both types, the active center is a substrate cleft. S. also have a broader substrate specificity than the pancreatic enzymes. This is a notable example of the convergent evolution of catalytic activity in two structurally completely different classes of proteins. S. is used in the structural elucidation... 

Batistic et al. have used similar extraction and purification procedures to obtain from soil active acid and alkaline phosphomonoesterases (and other hydrolases, cellulase, B-glucosidase, invertase and proteinase), Salmine substituted for protamine as a precipitant of humic material from dialysed extracts. In this case complete flocculation of humic compounds was accompanied by extensive (about 60%) losses of enzymic activities from solution. The activities of the precipitated material were low and could not be restored. Enzymes remaining in dialysed supernatants were of enhanced (about ten fold) specific activities, and were further fractionated by... 

During the course of our earlier studies on the conversion of fructose 1,6-bisphosphatase (Fru-Pgase) from the neutral to the alkaline form by lysosomal proteinases from rabbit liver (14-16) we observed that a considerable fraction of the converting enzyme activity, designated CE-II (15), remained associated with the insoluble membrane fraction after disruption of the lysosomes by freezing and thawing. This membrane-bound activity was found to be almost fully expressed by undisrupted rabbit liver lysosomes (15, 17). Rabbit liver cathepsin B, which also exhibited Fru-Paase-converting enzyme activity (15, 17, 18), was not present in the membrane fraction and was not responsible for the activity observed with intact lysosomes (17). 

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