Presence Of Diisopropylfluorophosphate Research Articles

Degradation of neocarzinostatin by blood sera in vitro and its inhibition by diisopropyl fluorophosphate and n-ethylmaleimide.

Inactivation of antitumor antibiotic, Neocarzinostatin, which occurs in vivo or during incubation with serum in a test tube, was found to depend on the dose of serum and incubation period. The inactivation process was accompanied with degradation of Neocarzinostatin to a smaller molecular size with concomitant loss in antigenic activities at a slower rate. Apparent increase, close to about 250% of control, in the antibiotic activity was found before the inactivation, in the presence of diisopropyl fluorophosphate, or about 200% in the presence of N-ethylmaleimide. The present findings suggest that the degradation process partly depends on proteolysis by serine type proteinase(s) and partly on the involvement of sulfhydryl groups. These results imply two possible applications; one for chemotherapy with Neocarzinostatin in combination with inhibitors of proteolysis and the other for drug design through the modification of amino acid residues on Neocarzinostatin molecules which provide the enzyme binding sites such as lysine and arginine for the proteolytic catalysis.

The Conversion of Prothrombin to Thrombin: II. DIFFERENTIATION BETWEEN THROMBIN- AND FACTOR Xa-CATALYZED PROTEOLYSES

Abstract Prothrombin is activated by activated Factor X ± (Factor V and phospholipid) in the presence of diisopropylfluorophosphate which preferentially inhibits thrombin. Under these conditions a single activation fragment, designated Fragment 1·2, is released which represents the complete nonthrombin portion of the prothrombin molecule. The same fragment is seen as a transient species in the absence of diisopropylfluorophosphate when the activation reaction is monitored by disc electrophoresis in 8 m urea. The NH2terminal residue of both prothrombin and Fragment 1·2 is alanine, suggesting that Fragment 1·2 is derived from the NH2-terminal end of the prothrombin molecule. Fragment 1·2 is not cleaved by activated Factor X ± (Factor V and phospholipid), but is cleaved by thrombin to form two fragments which are identical by electrophoresis, ion exchange chromatography, and amino acid composition to Fragment 1, the fragment released from prothrombin on incubation with thrombin, and Fragment 2, the fragment released by activated Factor X when Intermediate 1 is converted to Intermediate 2 (Paper I; Owen, W. G., Esmon, C. T., and Jackson, C. M. (1974) J. Biol. Chem. 249, 594–605). It is proposed that the formation of Intermediate 1 during prothrombin activation is solely the result of thrombin-catalyzed proteolysis of prothrombin.

Activation of prothrombin with (taipan snake) venom

Complete activation of prothrombin with taipan snake () venom yields thrombin and two fragments, fragment-1 and fragment-2, which by the criteria of ion-exchange chromatography and SDS gel electrophoresis are indistinguishable from the products of Xa-catalyzed prothrombin activation. Activation in the presence of diisopropylfluorophosphate with either taipan venom or Xa results in the formation of thrombin-sensitive fragment 1·2, a single polypeptide composed of fragments-1 and 2. Whereas the rate of venom-catalyzed activation is enhanced 20–30 times by phospholipid vesicles, Factor V is without effect in either the presence or absence of phospholipid. The venom activity is not inhibited by diisopropylfluorophosphate, phenylmethylsulfonylfluoride, soybean trypsin inhibitor, p-hydroxymercuribenzoate or iodoacetate. The venom does not hydrolyze N2-toluene-p-sulfonyl-L-arginine methyl ester, N2-benzoyl-L-arginine ethyl ester or N-acetyl-L-tyrosine ethyl ester. The rate of venom-catalyzed prothrombin activation is not affected by plasma factor Xa inhibitor plus heparin.

Evidence for Proteolytic Degradation of Histidinol Phosphate Phosphatase Specified by Nonsense Mutants of the hisB Gene of Salmonella typhimurium

The gene products from hisB nonsense mutants having histidinol phosphate phosphatase activity were isolated from Salmonella typhimurium. The enzyme from strain TR691 (hisB278 hisT1529 aroD5) was isolated in the presence of diisopropylfluorophosphate. Three electrophoretically separable forms were demonstrated, and all were shown to have a mol wt of approximately 38,000 and to consist of a single polypeptide chain. Previously, two forms of the phosphatase enzyme from this strain were isolated without diisopropylfluorophosphate and shown to have a different subunit composition. Strain TA387 (hisB2133 his 01242) was shown to have two electrophoretically separable phosphatases with a mol wt of about 52,000 and consisted of 17,000- to 19,000-mol wt polypeptide chains as evidenced by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The results could be explained by proteolytic cleavage of the primary gene product which can partially be prevented by the protease inhibitor. Strain TA387 phosphatase lost all activity in 8 M urea but could be renatured by dialysis. Gel filtration showed that it also regained its original molecular weight. The values of K(m) of histidinol phosphate and the competition inhibition constant for histidinol were determined. The addition of MnCl(2) to the assay was shown to shift the optimal pH value to a lower pH value.

Purification of blood coagulation factors II, VII, IX and X from bovine citrated plasma.

The bovine coagulation factors II, VII, IX and X were separated from other plasma proteins, and each factor was obtained in a largely pure state by various chromatographic procedures in the presence of diisopropylfluorophosphate (DFP). Factor X was separated from factors II, VII, and IX by DEAE Sephadex chromatography, followed by G-100 filtration and hydroxylapatite chromatography. Separation of factors II and IX from factor VII was achieved by chromatography on DEAE cellulose, followed by G-100 gel filtration and hydroxylapatite chromatography. Factors II and IX were separated by preparative polyacrylamide electrophoresis. After DEAE cellulose chromatography, factor VII was purified by gradient elution from hydroxylapatite.

Lipase of Mucor pusillus.

Lipase of Mucor pusillus NRRL 2543 was recovered with ammonium sulfate precipitation, gel filtration on Sephadex G-75, and anion-exchange chromatography on diethylaminoethyl-Sephadex A-50. Maximal glycerol ester hydrolase (lipase) activity was observed at pH 5.0 to 5.5 and 50 C when trioctanoin and olive oil were used as substrates. The enzyme also showed esterase activity; it hydrolyzed, with the exception of methyl butyrate, all methyl esters tested. A minimum chain length of six carbons appeared to be a requirement for esterase activity, which was maximal at about pH 5.5 with methyl dodecanoate (C(12)) as the substrate. Neither the glycerol ester hydrolase (lipase) nor the esterase activity of the enzyme appeared to be affected by thiol group inhibitors, chelating agents, and reducing compounds. On the other hand, hydrolysis of triolein and methyl dodecanoate was arrested to the same extent in the presence of diisopropyl fluorophosphate, which suggested the involvement of serine in the active center of the enzyme. The enzyme remained stable during a 30-day storage at - 10 C.

Lipase of Mucor pusillus1

Lipase of Mucor pusillus NRRL 2543 was recovered with ammonium sulfate precipitation, gel filtration on Sephadex G-75, and anion-exchange chromatography on diethylaminoethyl-Sephadex A-50. Maximal glycerol ester hydrolase (lipase) activity was observed at pH 5.0 to 5.5 and 50 C when trioctanoin and olive oil were used as substrates. The enzyme also showed esterase activity; it hydrolyzed, with the exception of methyl butyrate, all methyl esters tested. A minimum chain length of six carbons appeared to be a requirement for esterase activity, which was maximal at about pH 5.5 with methyl dodecanoate (C(12)) as the substrate. Neither the glycerol ester hydrolase (lipase) nor the esterase activity of the enzyme appeared to be affected by thiol group inhibitors, chelating agents, and reducing compounds. On the other hand, hydrolysis of triolein and methyl dodecanoate was arrested to the same extent in the presence of diisopropyl fluorophosphate, which suggested the involvement of serine in the active center of the enzyme. The enzyme remained stable during a 30-day storage at - 10 C.

Implication of an Ionizing Group in the Control of Conformation and Activity of Chymotrypsin

Abstract 1. Acetylated chymotrypsinogen was prepared by the reaction of crystallized chymotrypsinogen with acetic anhydride at pH 6.7 and 4°, and purification of the product. End group analysis and Van Slyke amino nitrogen determination indicated that all the free e- and α-amino groups of the chymotrypsinogen molecule had been blocked by acetylation. 2. Activation of acetylated chymotrypsinogen by trypsin, followed by chromatography, yielded acetylated δ-chymotrypsin. Quantitative end group determination indicated that the molecule had a single amino group, the NH2-terminal α-amino group of an isoleucine residue. In the catalytic hydrolysis of N-acetyl-l-tryptophan amide, the steady state kinetic parameters were found to be the same, in the pH region 6 to 10, with acetylated δ-chymotrypsin and with δ-chymotrypsin. 3. The activation of acetylated chymotrypsinogen to acetylated δ-chymotrypsin was found to be accompanied by the appearance of a single new ionizing group, titratable in the pH region 5 to 10.5, with a pK(app) of 8.3. 4. The specific rotation of acetylated δ-chymotrypsin was found to vary with pH in the pH region 6 to 10.5, the change in specific rotation following the ionization of a single group with pK(app) of 8.3. Above pH 10 the specific rotation of the zymogen and the enzyme were found to be the same at the three different wave lengths investigated. 5. Previous investigations in this and other laboratories of the pH dependence of the quotient of the steady state kinetic parameters, kcat [Km(app)]-1 —a function which yields the ionization constants of the catalytically importance ionizing groups of the free enzyme—have indicated the importance of an ionizing group with pK(app) ∼ 8.5 in all α-chymotrypsin-catalyzed reactions studied, and in the δ- or acetylated δ-chymotrypsin-catalyzed hydrolysis of N-acetyl-l-tryptophan amide. 6. An interpretation of these results that is consistent with all available experimental information is that the conversion of catalytically inactive chymotrypsinogen to active enzyme is accompanied by the appearance of a new ionizing group (a group that has a pK(app) of 8.3 in acetylated δ-chymotrypsin) which controls the conformation and thereby the activity of the enzyme. Molecules in which this ionizing group is unprotonated are in a catalytically inactive conformation, resembling that of chymotrypsinogen, and molecules in which this ionizing group is protonated are in a catalytically active conformation. A diisopropyl fluorophosphate (DFP)-induced protein isomerization involves only the enzyme molecules that are in the catalytically active conformation. This interpretation leads to a prediction that in the presence of DFP there will be an increase in pK(app) of the ionizing group. Such an increase in pK(app) was previously observed, and accounts for the proton uptake and the change in specific rotation of the enzyme observed in its reaction with DFP. 7. All evidence obtained so far suggests that the group with pK(app) ∼8.5 is an NH2-terminal isoleucyl α-amino group.

Amino terminal amino acids of hog pancreatic amylase and their relationship to its enzymic activity

By reaction with 1-fluoro-2,4-dinitrobenzene (FDNB), hog pancreatic amylase was found to have three NH 2-terminal amino acids: phenylalanine, alanine, and glycine. In this reaction, FDNB reacted with only 12 out of 17 lysines and none of the histidine or tyrosine residues in the amylase. This may indicate a relatively compact structure for the amylase. Reaction of the NH 2-terminal amino acids with FDNB led to a large loss in activity with only one substituted and complete loss of activity when all three were reacted. Reaction of hog pancreatic amylase with O-methylisourea resulted in a 25% loss in enzyme activity with 25% of the α-amino groups and 65% of the ϵ-amino groups guanidated. The amylase used in these reactions was stabilized by the presence of diisopropylfluorophosphate, p-hydroxymercuribenzoate, iodoacetic acid, iodoacetamide, and calcium. Amylase stored without these protease inhibitors lost considerable activity but in their presence remained stable in the cold for months.