Showing papers on "Proteolytic enzymes published in 1970"

Proteolytic Enzymes Initiating Cell Division and Escape from Contact Inhibition of Growth

Abstract: BRIEF treatment with very low concentrations of proteolytic enzymes can bring about a change in the cell surface similar to that occurring in the chemical or viral transformation of normal to malignant cells1. This suggests to us that proteolytic enzymes may not only convert the surface structure into the type seen in malignant cells but that the whole cell may respond to the proteolytic enzyme with initiation of new rounds of cell division and a concomitant escape from contact inhibition of growth, as is the case for malignant cells in tissue culture2.

Mapping the Active Site of Papain with the Aid of Peptide Substrates and Inhibitors

Abstract: The active site of an enzyme performs the twofold function of binding a substrate and catalysing a reaction. The efficiency of these actions determines the overall activity of the enzyme towards the particular substrate, i.e. determines the specificity of the enzyme. It is therefore possible to obtain information on the active site by the kinetics of the enzyme’s reactions with different substrates and inhibitors. An important feature of the active site is its size. It should be possible to 'measure’ this by using substrates or inhibitors large enough to show up the interactions of the furthermost parts of the binding site. In the present series of investigations on proteolytic enzymes, our approach is to compare the activity of the enzyme towards ( a ) peptides of increasing length, ( b ) diastereoisomeric pairs of peptides in which a particular amino acid residue has been replaced by its antipode, and ( c ) pairs of substrates in which a particular side chain (say a methyl group) has been replaced by another (say an aromatic group). The influence of these changes on reaction rates as a function of distance from the point of cleavage indicates the extent of the active site (Schechter, Abramowitz & Berger 1965; Abramowitz, Schechter & Berger 1967).

Release from Density Dependent Growth Inhibition by Proteolytic Enzymes

Abstract: CONFLUENT chick embryo cells in culture grow much more slowly than do sparse cells in conditions where significant depletion of the medium is avoided1–3. Because an important factor regulating growth seems to be the population density, this phenomenon is called density dependent inhibition of growth4. When a thin scratch is made in a slowly growing confluent culture of chick embryo cells, the cells adjoining the scratch begin to grow rapidly and continue to do so until confluence is restored3. Growth of confluent cells can also be stimulated by the addition of a macromolecular component of serum5. We now report that the addition of proteolytic enzymes to the medium of inhibited cells in concentrations too low to cause cell detachment stimulates rapid growth among the cells.

Characterization of proteinases excreted by Bacillus subtilis Marburg strain during sporulation.

Abstract: Summary. This study has characterized 3 proteolytic enzymes during sporulation by Bacillus subtilis Marburg strain when grown in nutrient broth. A method of purification is described which permits the separation of 2 different proteinases: one belonging to the metal enzyme group and the other to the serine enzyme group. The third enzyme, probably an esterase, showed a high esterolytic activity, but only low proteolytic activity. Determination of the 3 enzymes in a mixture was accomplished by using specific substrates and inhibitors. They were excreted simultaneously between the end of the growth phase until the appearance of the prespores. During this entire period, 20% of the total proteolytic activity was due to the metal proteinase; 80% of the proteolytic activity and 15% of the esterolytic activity was due to the serine proteinase; 85% of the esterolytic activity was the result of the esterase. These findings will contribute to a more complete phenotypic characterization of those mutants of sporulation that appear to be involved in the production of extracellular hydrolytic enzymes.

Specific glucocorticoid-binding macromolecules from mouse fibroblasts growing in vitro. A possible steroid receptor for growth inhibition.

Abstract: Mouse fibroblasts growing in vitro (L cells) contain a binding component for triamcinolone acetonide which is apparently distributed intracellularly, largely as a cytoplasmic soluble macromolecule. The structure-activity relationships of steroids active in growth inhibition are exactly paralleled by their ability to displace 3H-triamcinolone acetonide from this binding component. The binding component is saturated between 10-8 and 5 x 10-8 M triamcinolone acetonide. The macromolecules bind unchanged triamcinolone acetonide noncovalently, and the steroid is released from binding by brief digestion with a proteolytic enzyme. Cells which are resistant to glucocorticoids bind much less triamcinolone acetonide in a specific manner (i.e., displaceable by glucocorticoids) than do sensitive cells. The binding component therefore satisfies many of the rigorous criteria necessary for assignment as a "receptor" for the growth-inhibitory action of glucocorticoids on mouse fibroblasts. In addition, triamcinolone acetonide bound to the 105,000 x g supernatant fraction remains bound under conditions of frozen storage for at least 1 month.

The composition of milk xanthine oxidase

Abstract: The composition of milk xanthine oxidase has been reinvestigated. When the enzyme is prepared by methods that include a selective denaturation step in the presence of sodium salicylate the product is obtained very conveniently and in high yield, and is homogeneous in the ultracentrifuge and in recycling gel filtration. It has specific activity higher than previously reported preparations of the enzyme and its composition approximates closely to 2mol of FAD, 2g-atoms of Mo and 8g-atoms of Fe/mol of protein (molecular weight about 275000). In contrast, when purely conventional preparative methods are used the product is also homogeneous by the above criteria but has a lower specific activity and is generally comparable to the crystallized enzyme described previously. Such samples also contain 2mol of FAD/mol of protein but they have lower contents of Mo (e.g. 1.2g-atom/mol). Amino acid compositions for the two types of preparation are indistinguishable. These results confirm the previous conclusion that conventional methods give mixtures of xanthine oxidase with an inactive modification of the enzyme now termed ;de-molybdo-xanthine oxidase', and show that salicylate can selectively denature the latter. The origin of de-molybdo-xanthine oxidase was investigated. FAD/Mo ratios show that it is present not only in enzyme purified by conventional methods but also in ;milk microsomes' (Bailie & Morton, 1958) and in enzyme samples prepared without proteolytic digestion. We conclude that it is secreted by cows together with the active enzyme and we discuss its occurrence in the preparations of other workers. Studies on the milks of individual cows show that nutritional rather than genetic factors determine the relative amounts of xanthine oxidase and de-molybdo-xanthine oxidase. A second inactive modification of the enzyme, now termed ;inactivated xanthine oxidase', causes variability in activity relative to E(450) or to Mo content and formation of it decreases these ratios during storage of enzyme samples including samples free from demolybdo-xanthine oxidase. We conclude that even the best purified xanthine oxidase samples described here and by other workers are contaminated by significant amounts of the inactivated form. This may complicate the interpretation of changes in the enzyme taking place during the slow phase of reduction by substrates. Attempts to remove iron from the enzyme by published methods were not successful.

Three-dimensional Fourier synthesis of tosyl-elastase at 3.5 å resolution.

Abstract: X-ray diffraction data have been collected from four isomorphous crystalline derivatives of the proteolytic enzyme elastase, and a map of the three-dimensional electron density of the tosyl derivative has been calculated by Fourier synthesis of 2,750 independent terms. It shows clearly the conformation of the elastase polypeptide chain, the size and orientation of the amino-acid side-chains, and the position of the tosyl group which labels the active centre of the enzyme.

Survey of workers exposed to dusts containing derivatives of Bacillus subtilis.

Abstract: In a survey of 121 workers exposed to dusts containing derivatives of Bacillus subtilis, mainly proteolytic enzymes, skin tests showed evidence of sentiztation was higher among “atopic” subjects—16 out of tization was higher among “atopic” subjects—16 out of 25 (64%)—than among “non-atopic” subjects—32 out of 96 (33%). Reduced ventilatory capacity was found in 44% of sensitized workers compared with 14% of those not sensitized.

Synthesis and Cleavage of Enterovirus Polypeptides in Mammalian Cells

Abstract: Evidence is presented that the entire enterovirus ribonucleic acid genome is translated into a single giant polypeptide of well over 200,000 daltons molecular weight. This giant protein was detected repeatedly in coxsackievirus B1-infected cells, even in the absence of amino acid analogues. The enzymes which cleave this giant protein into smaller structural and enzymatic proteins accumulate with increasing time after infection. It appears that they are either virus-coded proteolytic enzymes or else host enzymes which are activated or released from an intracellular site as a result of virus infection. These cleavage enzymes do not cause grossly apparent cleavage of host-cell proteins.

The effect of trypsin inhibitors on pancreatopeptidase E, trypsin, chymotrypsin and amylase in the pancreas and intestinal tract of chicks receiving raw and heated soya-bean diets.

Abstract: 1. Feeding on a raw soya-bean diet (RSD) increased the levels of trypsin, chymotrypsin and pancreatopeptidase E but decreased the level of amylase in the pancreas of chicks as compared to a heated soya-bean diet (HSD), while supplementation of HSD with soya-bean trypsin inhibitors increased the activity of all four enzymes. HSD + trypsin inhibitors caused significant enlargement of the pancreas but only a slight depression in growth rate.2. Fasting for 24 h of chicks previously given RSD and HSD increased the activity of all four enzymes but the increase was much greater in chicks previously given RSD than in those previously given HSD.3. Feeding RSD for 4 d to chicks previously adapted to HSD resulted in a dramatic inhibition in growth rate, a small increase in pancreas weight, and an increase in the activity of all proteolytic enzymes, while no change in the amylase was detectable.4. Trypsin, chymotrypsin and pancreatopeptidase E activities were assayed in the contents of the small intestine and caecum of chicks fed on RSD or HSD over a period of 35 d. Trypsin and chymotrypsin activities in the small intestine were lower in chicks fed on RSD while pancreatopeptidase E activity was almost equal or even higher in RSD-fed chicks, especially at the age of 35 d. Trypsin activity in the caecum of RSD-fed chicks was lower at all stages of the experiment, while the pancreatopeptidase E and chymotrypsin activities in the caecum of RSD-fed chicks exceeded the levels in the HSD group at the age of 21 and 35 d respectively. It would appear therefore that pancreatopeptidase E may play an important part in overcoming the inhibition of the proteolytic activity in the intestine of chicks fed on RSD.

Enhancement of reovirus infectivity by extracellular removal or alteration of the virus capsid by proteolytic enzymes.

Abstract: Summary Reovirus particles have an inner coat between the capsid and the nucleic acid core. The in vitro removal of the capsid layer by proteolytic enzymes resulted in an increase in infectivity in reovirus preparations. This finding contributes to a better understanding of virus infection, stability and structure, and helps explain results of kinetic studies of activation and inactivation. Further, the findings presented have practical application in the isolation and identification of reovirus, and in the preparation of high-titred virus stocks.

The structure and mechanism of action of papain.

Abstract: The cysteine proteinases form a group of enzymes which depend for their enzymic activity on the thiol group of a cysteine residue. Several which occur in plants have been investigated extensively and include papain, ficin and stem bromelain (Smith & Kimmel i960). Although the term papain, introduced last century to describe the proteolytic principle in papaya latex (Wurtz & Bouchut 1879) is still used to describe crude dried latex, the crystalline enzyme is readily obtained (Kimmel & Smith 1954). Ficin is known to consist of several closely related enzymes which have been resolved (Sgarbieri, Gupte, Kramer & Whitaker 1964), but for most structural and mechanistic studies the unresolved mixture of enzymes has been used. Stem bromelain also appears to be a mixture of at least two proteolytic enzymes which have not yet been resolved (Ota, Moore & Stein 1962; Murachi 1964). In spite of the recognized heterogeneity of ficin and stem bromelain, it does seem that both structurally and mechanistically they are similar to papain. Only one bacterial cysteine proteinase has received a detailed study, namely, streptococcal proteinase, and it appears to have little or no relation in its amino acid sequence with the plant enzymes (Liu, Stein, Moore & Elliott 1965). The functional groups involved in the catalytic mechanism are apparently the same as in the plant proteinases (Gerwin, Stein & Moore 1966; Liu 1967; Husain & Lowe 1968 a , c ), but the mechanism of action has not been extensively studied. It may well be however that the plant and bacterial cysteine proteinases have converged onto a similar mechanism of action by two independent evolutionary pathways, as now seems apparent for the animal and bacterial serine proteinases (Alden, Wright & Kraut, this volume, p. 119). Because the tertiary crystal structure of papain (Drenth, Jansonius, Koekoek, Swen & Wolthers 1968; see also the preceding paper, p. 231) is now known, a critical survey of this enzyme is apposite.

Proteolytic enzymes of Saccharomyces carlsbergensis.

Abstract: 1. Of four proteolytic enzymes isolated from autolysing Saccharomyces carlsbergensis, one is inactivated at about 45°C, whereas the others are stable at 50°C. pH optima for activity are from 3.0 to 8.0 but maximum stability is between pH6.0 and 6.5. All appear to be glycoproteins, the carbohydrate moiety containing glucose and mannose residues. 2. Lysed protoplasts of the same yeast release four proteolytic enzymes each of which have two pH optima at pH3.0 and 7.0 approximately. Compared with the enzymes from autolysed yeast, resistance to high temperature is much less, and they are not glycoprotein in nature. 3. The same yeast grown with N-acetyltyrosine ethyl ester as nitrogen source secretes into the medium four proteases believed to be glycoprotein in nature. Generally they resemble the enzymes from lysed protoplasts more than those from autolysing yeast.

APPLYING PROTEOLYTIC ENZYMES ON SOYBEAN. 6. Deodorization Effect of Aspergillopeptidase A and Debittering Effect of Aspergillus Acid Carboxypeptidase

Abstract: SUMMARY— A proteolyzate obtained by treating an isolated soybean protein preparation with Molsin, a crude preparation of aspergillopeptidase A (APase A), was less bitter and contained larger amounts of free amino acids, especially hydrophobic amino acids. A proteolyzate obtained by treating this protein preparation with crystallized APase A was much more bitter and contained smailer amounts of free amino acids, mainly consisting of hydrophilic amino acids. The latter was richer in peptides than the former, bearing hydrophobic amino acid residues near the C-termini. Difference in N-terminal amino acid composition apparently has not been found between the 2 proteolyzates. These results indicate that Molsin per se contains a certain carboxypeptidase which decomposes the C-terminal structures and, consequently, lessens the bitterness (debittering effect). This carboxypeptidase was found to be identical with aspergillus acid carboxypeptidase (AACPase). Abase A, as well as MO/sin, was effective in removing odor ants, i.e., n-hexanal, n-hexanol and n-heptanol, from the isolated soybean protein preparation Ideodorization effect). AACPase seemed to have no deodorization effect. A method was suggested to prepare a deodorized and debittered proteolyzate by a combination use of APase A and AACPase.

The biochemical basis of the fungus-attine ant symbiosis.

Abstract: The natural history of the fungus-growing ants provides a spectacular example of a symbiotic association of two very different types of organisms. An anthropomorphic description is difficult to resist. The ants are efficient and industrious farmers. Their single crop is a fungus, grown on a substrate of leaves in carefully fertilized, welltended gardens. Virtually every facet of the ants' behavior and life cycle has been shaped by their association with the fungus they culture. A characteristic feature of the ants' gardening technique is the application of their fecal material to the garden and to substrate being prepared for incorporation into the garden. We have established the biochemical significance of this behavior. The fecal material contains proteolytic enzymes which compensate for a deficiency of such enzymes in the fungus. In addition, the nitrogenous components in the fecal material facilitate the initial growth of the fungus. In biochemical terms, then, one can say that the ants contribute their enzymatic apparatus to degrade protein and the fungus contributes its enzymatic apparatus to degrade cellulose. As in the case of so many other natural symbiotic and parasitic associations, the basis is an integration of complementary metabolic capabilities and deficiencies.