Reaction of Ficin with Diisopropylphosphorofluoridate. Evidence for a Contaminating Inhibitor<sup>*</sup>

Gould, N. R.; Liener, Irvin E.

doi:10.1021/bi00877a016

Cited by 43 publications

(23 citation statements)

References 33 publications

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“…(a) The reaction of DFP with protein is not limited to the active site of serine enzymes; DFP can form stable derivatives with the hydroxyl group of tyrosine residues, and with serine and/or threonine hydroxyls that are not located in enzymatically active centers (14)(15)(16)(17). 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.…”

Section: Discussionmentioning

confidence: 99%

“…1 b and legend to Fig. 1) makes it seem likely that at least some of the incorporated DFP was due to reaction with these trypsin-like enzymes, but the fractional labeling represented by such enzymes could not be estimated from these observations alone, as DFP can bind covalently to proteins other than those containing active serine groups (14)(15)(16)(17). Accordingly, the nature of the [3H]DFP reactive proteins was characterized further by performing the labeling reaction in the presence of a variety of known, competing, protease inhibitors.…”

Section: Analysis Of Conditioned Media From Cultures Of Normal and Trmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

Section: Analysis Of Conditioned Media From Cultures Of Normal and Trmentioning

confidence: 99%

Serine enzymes released by cultured neoplastic cells.

Danø¹,

Reich²

1978

The Journal of Experimental Medicine

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“…To determine whether cancer procoagulant was inhibited by DFP or by the conjectured impurity of DFP, samples of DFP were treated with cupric ion and imidazole to catalyze the hydrolysis of DFP (17) and tested for their inhibitory capacity at various times during the hydrolysis reaction (18). A solution of25 mM CuSO4 and 25 mM imidazole was prepared in water, adjusted to pH 7.6 with 1 N NaOH, and made 10 mM DFP by the addition of 0.5 M DFP in isopropanol.…”

Section: Introductionmentioning

confidence: 99%

“…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%

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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“…Solutions were diluted-2 fold with enzyme and incubated 18 h at 4 C prior to being assayed. DFP was diluted to 10 mm or 2 mm in 50 mm Na-phosphate, 20 mm Cys (pH 7.0), and preincubated for 30 min at ambient temperature to reduce oxidants in DFP known to be inhibitory to enzymes (13). DFP solutions were then diluted 2-fold with enzyme solutions and incubated for 1 h at 25 C before assay.…”

Section: Methodsmentioning

confidence: 99%

Peptidohydrolases of Soybean Root Nodules

Malik

Pfeiffer²,

Williams³

et al. 1981

Plant Physiol.

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Nodule extracts prepared from Glycine max var Woodworth possessed endopeptidase, aminoptidase, and carboxypeptidase actiities. Three distinct endopeptidase activities could be resolved by disc-gel electrophoresis at pH 8.8. According to their order of increasing electrophoretic mobility, the first of these enzymes hydrolyzed azocasein and n-benzoyl-LLeu-p8-naphthylamide, while the second hydrolyzed n-benzoyl-L-Arg-/8-, and azocasein. The third endopeptidase hydrolyzed Bz-L-Arg-8NA, Bz-L-Arg-pNA, and hemoglobin. Fractions of these enzymes extracted from electrophoresis gels were shown to have pH optima from 7.5 to 9.8.AU of the endopeptidases were completely inhibited by diisopropylphosphorofluoridate, demonstrating that they were serine proteases.Aminopeptidase activity was measured using amino acyl-p-naphthylamides. Electrophoresis of nodule extracts at pH 6.8 resolved the aminopeptidase activity of nodule extracts into at least four fractions based on mobility and on activities toward amino acyl-pi-naphthylamides. The major activity of two of the aminopeptidases was directed toward L-Leu-and LMet-p-naphthylamide, while the other two aminopeptidases exhibited broader specificity and were capable of hydrolyzing a large number of amino acyl-.-naphthylamides. Two of the aminopeptidases extracted from electrophoresis gels were classiffed as thiol type enzymes, and all four aminopeptidases had neutral to basic pH optima.Senescence of soybean root nodules results in a decreased capacity to reduce atmospheric nitrogen, thus potentially limiting the supply of amino nitrogen to the plant. After the decline in nitrogenase activity, total soluble protein per unit fresh weight of nodule decreases rapidly (22), although the fresh weight per nodule shows little change. In addition, the total number of nodules per plant decreases sharply. Thus, nodule senescence can be characterized by a sharp decline in nitrogenase activity coupled with a degeneration of the nodule leading to loss of soluble protein and eventual abscission of the nodule from the root. The loss of soluble protein is a characteristic feature of all types of plant senescence, and is generally attributed to increased activity of proteolytic enzymes. The role of proteolytic enzymes in nodule senescence is not as well established as in leaf senescence (7, 8, 19, '

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Reaction of Ficin with Diisopropylphosphorofluoridate. Evidence for a Contaminating Inhibitor^*

Cited by 43 publications

References 33 publications

Serine enzymes released by cultured neoplastic cells.

Serine enzymes released by cultured neoplastic cells.

A factor X-activating cysteine protease from malignant tissue.

Peptidohydrolases of Soybean Root Nodules

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Reaction of Ficin with Diisopropylphosphorofluoridate. Evidence for a Contaminating Inhibitor*

Cited by 43 publications

References 33 publications

Serine enzymes released by cultured neoplastic cells.

Serine enzymes released by cultured neoplastic cells.

A factor X-activating cysteine protease from malignant tissue.

Peptidohydrolases of Soybean Root Nodules

Contact Info

Product

Resources

About

Reaction of Ficin with Diisopropylphosphorofluoridate. Evidence for a Contaminating Inhibitor^*