Alkylphosphorylation of Stem Bromelain by Diisopropylphosphorofluoridate without Inhibition of Proteinase Activity<sup>*</sup>

Murachi, Takashi; Yasui, Mieko

doi:10.1021/bi00887a003

Cited by 31 publications

(26 citation statements)

References 24 publications

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“…Although there is disagreement, the studies generally conclude that commercial DFP inhibits cysteine proteases but that the inhibition of the active site sulfhydryl is due to an undefined impurity in most commercial preparations. Purified DFP phosphorylates serine (18,35) (18,(30)(31)(32)35), and the DFP impurity irreversibly inhibits the active site sulfhydryl (18,31). Since the evidence presented here suggested cancer procoagulant was a cysteine protease, it seemed likely that the inhibition by DFP, a consistent and reproducible observation (9,10,14), was probably due to impurities in the commercial DFP preparations that were employed.…”

Section: Introductionsupporting

confidence: 53%

“…Although other investigators suggest that inhibition of cysteine proteases by the DFP impurity is essentially irreversible (18,29,31), their data show partial (-10%) reactivation of bromelain with 5 mM cysteine (29) and about 40% reactivation of ficin with 0.25 M cysteine (18). Our use of DTT, a more potent reducing agent than cysteine, different reactivation conditions, and the differences in the cysteine proteases may account for our more effective reactivation of DFPinhibited cancer procoagulant.…”

Section: Introductionmentioning

confidence: 74%

“…DFP and PMSF are well-known irreversible inhibitors of serine proteases (20,26,27); PMSF is a less effective inhibitor of cysteine proteases, and is reversible (20,21,28); and the DFP inhibition of cysteine proteases is controversial, as discussed below. Iodoacetamide and mercury are more specific inhibitors for cysteine proteases and are less effective or even ineffective for the inhibition of serine proteases (20)(21)(22) (29)(30)(31), ficin (18), papain (32,33), and chymopapain (34,35). Although there is disagreement, the studies generally conclude that commercial DFP inhibits cysteine proteases but that the inhibition of the active site sulfhydryl is due to an undefined impurity in most commercial preparations.…”

Section: Introductionmentioning

confidence: 99%

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A factor X-activating cysteine protease from malignant tissue.

Gordon¹,

Cross²

1981

J. Clin. Invest.

224

109

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A B S T R A C T A proteolytic procoagulant has been identified in extracts of human and animal tumors and in cultured malignant cells. It directly activated Factor X but its similarity to other Factor X-activating serine proteases was not clear. This study describes work done to determine whether this enzyme, cancer procoagulant, is a serine or cysteine protease. Purified cancer procoagulant from rabbit V2 carcinoma was bound to a p-chloromercurialbenzoate-agarose affinity column and was eluted with dithiothreitol. The initiation of recalcified, citrated plasma coagulation activity by cancer procoagulant was inhibited by 5 mM diisopropylfluorophosphate, 1 mM phenylmethylsulfonylfluoride, 0.1 mM HgCl2, and 1 mM iodoacetamide. Activity was restored in the diisopropylfluorophosphate-, phenylmethylsulfonylfluoride-, and HgCl2-inhibited samples by 5 mM dithiothreitol; iodoacetamide inhibition was irreversible. Russell's viper venom, a control Factor Xactivating serine protease, was not inhibited by either 0.1 mM HgCl2 or 1 mM iodoacetamide. The direct activation ofFactor X by cancer procoagulant in a two-stage assay was inhibited by diisopropylfluorophosphate and iodoacetamide. Diisopropylfluorophosphate inhibits serine proteases, and an undefined impurity in most commercial preparations inhibits cysteine proteases. Hydrolysis of diisopropylfluorophosphate with CuS04 and imidazole virtually eliminated inhibition of thrombin, but cancer procoagulant inhibition remained complete, suggesting that cancer procoagulant was inhibited by the undefined impurity. These results suggest that cancer procoagulant is a cysteine endopeptidase, which distinguishes it from other coagulation factors including tissue factor. This and other data suggest that neoplastic cells produce this unique cysteine protease which may initiate blood coagulation.

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Section: Introductionsupporting

confidence: 53%

Section: Introductionmentioning

confidence: 74%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A factor X-activating cysteine protease from malignant tissue.

Gordon¹,

Cross²

1981

J. Clin. Invest.

224

109

View full text Add to dashboard Cite

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“…In spite of these shortcomings there is sound evidence to suggest that the incorporation of [3H]DFP can serve as a versatile and sensitive means for detecting and identifying serine enzymes, particularly serine proteases: thus, (1) both the rate and extent of nonspecific reactions are markedly reduced below pH 7.5 and at DFP concentrations below millimolar (14,20); hence the risk of nonspecific labeling with [3H]DFP can be minimized by selecting the reaction conditions appropriately and with the reactivity of particular enzymes in mind. (2) It appears likely that the value of [~H]DFP for labeling serine enzymes will be greatest when the reaction is used in conjunction with enzyme assays and other enzymatic probes, such as known inhibitors.…”

Section: Discussionmentioning

confidence: 99%

Serine enzymes released by cultured neoplastic cells.

Danø¹,

Reich²

1978

The Journal of Experimental Medicine

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Transformation of embryonic cells from various species by oncogenic viruses is associated with large increases in production of plasminogen activators, and similar enzymes are released by cultures and/or cell lines derived from human and animal neoplasms. These enzymes activate plasminogen by limited proteolysis, and several of them have been identified as serine proteases because they are irreversibly inactivated by low concentrations of the active site reagent diisopropylfluorophosphate (DFP, 1 1-6). The stability of the covalent bond formed by the incorporation of radioactive DFP into serine residues at active sites (7, 8) can be used to label both plasminogen activators and other serine enzymes; subsequent analysis of the reaction products by gel electrophoresis and autoradiography should yield an identifiable spectrum of labeled species that might provide insights about the secretory activities of cultured cells.In this paper we present the initial results of such an approach to the comparison of serine enzymes released by transformed embryonic cells and their normal counterparts. To differentiate between serine proteases and esterases, and to obtain information about substrate specificity, we have taken advantage of the fact that incorporation of DFP can be blocked by prior reaction of enzymes with other active site reagents. The combination of these reactions can be used as a general procedure for identification of individual proteases in impure mixtures and particularly so for enzymes that are present at concentrations too low to permit detection by more traditional methods. We have also applied these methods to conditioned medium from several cultured human cell strains of neoplastic origin with a view of comparing the serine enzymes secreted by a variety of independently-derived cell types. Materials and MethodsCell Culture and Virus Infection. Cell cultures were prepared and maintained essentially as described by Unkeless et al. (1) using disposable plastic Petri dishes (100 mm diameter, Falcon Plastics, Division of BioQuest, Oxnard, Calif.) at 37°C in atmospheric air supplemented with 5% *

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“…Recently, Gould et al 8 ,91 and Murachi et al 10 ,IlI clarified that the inhibitions of sulfhydryl enzymes caused by DFP were due to impurities contaminated in commercial samples of DFP. Although, L-cysteine could protect the enzymes from DFP inhibition in the case of plant prateinases, the reverse was the case of yeast proteinase C. Further studies about the inhibitions caused by DFP and PCMB are now in progress.…”

Section: -Ilimentioning

confidence: 99%

Purification of Yeast Proteinases

Hata¹,

Doi²

1967

Agricultural and Biological Chemistry

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Three types of proteinases contained in baker's yeast were fractionated and partially purified by chromatography on TEAE-cellulose and on DEAE-Sephadex. These enzymes were designated as proteinase A, Band C, and some properties of proteinase A and B were described.A was similar to pepsin and "acid-proteinase" produced by various fungi, but did not hydrolyze carbobenzoxY-L-glutamyl-L-tyrosine. B had milk-clotting activity and proteolytic activity toward casein, and also esterolytic activity toward N-acetyl-L-tyrosine ethylester and a-N-benzoyl-L-arginine ethylester. This enzyme was inhibited by p-mercuribenzoate and diisopropylphosphorofluoridate. INTRODUCTIONIt has long been known that some proteinases and peptidases are contained in yeast cells and can be liberated after the cells are broken by autolysis. * This investigation was supported in part by a grant-in-aid for the scientific research from the Ministry of Education. This report was presented at the Annual Meeting of the Agricultural Chemical Society of Japan, Sapporo, July, 1964. I) G. Harris, "The Chemistry and Biology of Yeasts" edited by A. H. Cook, Academic Press, 1958, p. 437. 2) R. Willstiitter and W. Grassmann, Z. physiol. Chem., 153, 250 (1926).3) W. Grassmann and W. Haag, ibid., 167, 188 {1927). 4) W. Grassmann and H. Dyckerhoff, ibid., 179 41 (1928). ' 5) M. J. Johnson, J. Biol. Chem., 137, 575 (1940).6) F. Fehx and J. Labouesse-Mercouroff, Biochim. Biophys. Acta, 21, 303 (1956). 7) K. G. Demby, Biochem. Z., 81, 107 (1917).as "Hefetryptase" and "Hefepepsin", with optimum pH at 7.0 and 4.5, respectively. According to Grassmann et al.,"-4l yeast contained only one proteinase similar to papain, which was most active to many proteins at pH 5.0. Thereafter, Hecht and Civin Bl described that pepsin-like proteinase acting at pH 1.8 was present in the yeast autolysate. Recently, Lenney9l proved that the yeast proteinase consisted of two enzymes, measuring the activity by the method of Kunitz. l1l He reported that the two proteinases, A and B, were liberated by the autolysis of four different strains of Saccharomyces cerevisiae with chloroform. A exhibited an optimal pH of 3.7 for acid-denatured hemoglobin and was extremely labile in urea solution. On the other hand, B exhibited an optimal pH of 6.2 on urea-denatured proteins and was stable in urea solution and inhibited by sulfhydryl reagents. But purification and further characterization of these enzymes have not yet been performed by any authors. 8) M. Hecht and H. Civin, J. Biol. Chem., 116, 477 (1936). 9) J. F. Lenney, ibid., 221, 919 (1956).Purification of Yeast Proteinases. Part I 151In the present series of studies,lOI proteinases 'Of yeast have been fractionated by chromatography on TEAE-cellulose and DEAE-Sephadex columns, and separated into three enzymes which were designated as yeast proteinase A, Band C. MATERIALS AND METHODSMaterials. Compressed baker's yeast was obtained from Oriental Yeast Company. Hammarsten's casein was purchased from E. Merck AG., Darmstadt. Hemoglobin w...

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Alkylphosphorylation of Stem Bromelain by Diisopropylphosphorofluoridate without Inhibition of Proteinase Activity^*

Cited by 31 publications

References 24 publications

A factor X-activating cysteine protease from malignant tissue.

A factor X-activating cysteine protease from malignant tissue.

Serine enzymes released by cultured neoplastic cells.

Purification of Yeast Proteinases

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Alkylphosphorylation of Stem Bromelain by Diisopropylphosphorofluoridate without Inhibition of Proteinase Activity*

Cited by 31 publications

References 24 publications

A factor X-activating cysteine protease from malignant tissue.

A factor X-activating cysteine protease from malignant tissue.

Serine enzymes released by cultured neoplastic cells.

Purification of Yeast Proteinases

Contact Info

Product

Resources

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Alkylphosphorylation of Stem Bromelain by Diisopropylphosphorofluoridate without Inhibition of Proteinase Activity^*