scholarly journals Purification and reaction mechanism of the primary inhibitor of plasmin from human plasma

1978 ◽  
Vol 175 (2) ◽  
pp. 635-641 ◽  
Author(s):  
Ulla Christensen ◽  
Inge Clemmensen
Keyword(s):  
Dissociation Constant ◽  
Human Plasma ◽  
Active Site ◽  
Competitive Inhibition ◽  
Enzyme Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Inhibitory Effect ◽  
Exchange Chromatography ◽  
Comparison Of The Results ◽  
Sepharose 4B

The primary inhibitor of plasmin in human plasma was purified by a four-step procedure involving fractional (NH4)2SO4 precipitation, ion-exchange chromatography on a column of DEAE-Sepharose CL-6B and affinity chromatography on both a plasminogen–CH-Sepharose 4B column and a column of 6-aminohexanoic acid covalently coupled through the carboxylate function to AH-Sepharose 4B. No impurities in the final preparation could be detected when tested by immunoelectrophoresis against a range of specific antisera or against rabbit anti-human serum. On polyacrylamide-gel electrophoresis the inhibitor preparation showed a single band. The dissociation constant for the inhibitor–plasminogen complex was determined to be approx. 3μm at pH7.8. The reactions of the inhibitor with human plasmin and with bovine trypsin were studied. Comparison of the results obtained confirms the hypothesis previously presented, namely that the reaction of the inhibitor with plasmin involves at least two steps, the initial rapid formation of an enzyme–inhibitor complex followed by a slow irreversible transition to another complex. The results also indicate that the reaction of the inhibitor with trypsin involves just a single, irreversible step, so that this reaction seems to be less complicated than that of the inhibitor with plasmin. The ways in which 6-aminohexanoic acid influences the reactions were studied. The same value for the dissociation constant (approx. 26μm) for 6-aminohexanoic acid is obtained for both its effect on the reaction of the inhibitor with trypsin and for competitive inhibition of trypsin. The inhibitory effect of 6-aminohexanoic acid thus seems to be due to its blocking of the active site of trypsin. In contrast with this, the inhibitory effects of l-lysine and 6-aminohexanoic acid on the inhibitor–plasmin reaction occur at concentrations much too low to affect the active site of plasmin. The possible dependence of the reaction of the inhibitor with plasmin on a second site(s) on plasmin is discussed.

scholarly journals α-1-Anti-trypsin, an inhibitor of renin

1976 ◽  
Vol 153 (2) ◽  
pp. 505-507 ◽  
Author(s):  
S Scharpé ◽  
M Eid ◽  
W Cooreman ◽  
A Lauwers
Keyword(s):  
Human Plasma ◽  
Competitive Inhibition ◽  
Proteinase Inhibitors ◽  
Competitive Inhibitor ◽  
Inhibitory Effect ◽  
Renin Activity ◽  
Pig Kidney ◽  
Naturally Occurring ◽  
The Hill

A naturally occurring competitive inhibitor of pig kidney renin has been identified in human plasma. The inhibitor was shown to be α-1 anti-trypsin and the effect in vitro on the renin activity was examined. The slope in the Hill plot is compatible with the assumption of one-site competitive inhibition. Other proteinase inhibitors, such as α-2-macroglobulin and C1 inactivator, however, have no inhibitory effect on the renin-angiotensinogen reaction.

On the Interaction Between Human Plasma Kallikrein and C1-Esterase Inhibitor

1983 ◽  
Vol 49 (03) ◽  
pp. 193-195 ◽  
Author(s):  
Torbjörn Nilsson
Keyword(s):  
Dissociation Constant ◽  
Human Plasma ◽  
Rate Constant ◽  
Enzyme Inhibitor ◽  
Order Rate Constant ◽  
Second Order ◽  
Plasma Kallikrein ◽  
First Order ◽  
Order Rate ◽  
Esterase Inhibitor

SummaryThe kinetics of the reaction between human plasma kallikrein and CĪ-esterase inhibitor was studied in a purified system. By monitoring the inhibition reaction for extended periods of time, it was found to proceed in two consecutive steps, a fast reversible second-order binding step followed by a slower, irreversible first-order transition. The rate constants in this reaction model were determined, as well as the dissociation constant of the initial, reversible enzyme-inhibitor complex. Thus, at 37° C the second-order rate constant was found to be 5 · 104 M -1 · s-1, the first order rate constant was 5 · 10-4 s-1 and the dissociation constant K was 1.5 · 10-8 M. Heparin (28 U/ml) and 6-aminohexanoic acid (10 mM) had no effect on the k1 of the of the reaction.

scholarly journals T lymphocyte-derived differentiation-inducing factor inhibits proliferation of leukemic and normal hemopoietic cells

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1333-1338 ◽  
Author(s):  
U Gullberg ◽  
E Nilsson ◽  
MG Sarngadharan ◽  
I Olsson
Keyword(s):  
Cell Line ◽  
T Lymphocyte ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Inhibitory Effect ◽  
Exchange Chromatography ◽  
Leukemia Cells ◽  
Gel Chromatography ◽  
Wild Type ◽  
Clonogenic Growth

Abstract A differentiation-inducing factor (DIF) for the promyelocytic HL-60 cell line is constitutively produced by the malignant T lymphocyte line HUT-102. DIF was highly purified from HUT-102-conditioned media by means of diethylaminoethanol (DEAE)-chromatography, gel chromatography, and high-resolution, ion-exchange chromatography on a MonoQ column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition to inducing differentiation of wild-type HL-60 cells, resulting in secondary inhibition of growth, DIF, at a tenfold lower concentration, inhibited the growth of some clones of the monoblastic U- 937 cell line as well as that of subclones of HL-60. The latter effect was most likely a primary growth inhibition and not secondary to differentiation; 50% inhibition of clonogenic growth in agar was seen at approximately 1.0 pmol/L of DIF. In addition, the clonogenic growth of fresh leukemia cells from 10 of 12 patients with acute myeloid leukemia (AML) was inhibited with 50% inhibition at approximately 10 pmol/L of DIF. The growth of normal granulocyte-macrophage colonies was inhibited at a similar concentration, whereas early erythroid colonies were much more resistant. DIF and interferon-gamma (gamma-IFN) were shown to be separate molecules inasmuch as a neutralizing antibody for gamma-IFN did not abolish the DIF effect. The differentiation effect on wild-type HL-60 and the proliferation inhibitory effect on leukemic and normal myeloid cells cochromatographed through all purification steps suggest that both activities are exhibited by identical polypeptides. DIF may have a role in regulating normal hemopoiesis. The growth inhibitory effect of DIF and the ability to induce differentiation of some leukemia cells may suggest a clinical utility in the treatment of leukemia.

scholarly journals Inactivation of Δ5-3-oxo steroid isomerase with active-site-directed acetylenic steroids

1981 ◽  
Vol 193 (1) ◽  
pp. 217-227 ◽  
Author(s):  
T M Penning ◽  
D F Covey ◽  
P Talalay
Keyword(s):  
Active Site ◽  
Competitive Inhibition ◽  
Enzyme Inhibitor ◽  
Covalent Bond ◽  
Addition Product ◽  
Ring Structure ◽  
Dependent Manner ◽  
Compound I ◽  
Inhibitor Complex ◽  
Acetylenic Ketone

Several steroid analogues containing conjugated acetylenic ketone groups as part of a seco-ring structure or as substituents on the intact steroid system are irreversible inhibitors of delta 5-3-oxo steroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni. Thus 10 beta-(1-oxoprop-2-ynyl)oestr-4-ene-3,17-dione (I), 5,10-seco-oestr-4-yne-3,10,17-trione (II), 17 beta-hydroxy-5,10-seco-oestr-4-yne-3,10-dione (III) and 17 beta-(1-oxoprop-2-ynyl)androst-4-en-3-one (IV) irreversibly inactivate isomerase in a time-dependent manner. In all cases saturation kinetics are observed. Protection against inactivation is afforded by the powerful competitive inhibitor 19-nortestosterone. The inhibition constants (Ki) for 19-nortestosterone obtained from such experiments are in good agreement with those determined from conventional competitive-inhibition studies of enzyme activity. These compounds thus appear to be active-site directed. In every case the inactivated enzyme could be dialysed without return of activity, indicating that a stable covalent bond probably had formed between the steroid and enzyme. Compound (I) is a very potent inhibitor of isomerase [Ki = 66.0 microM and k+2 = 12.5 × 10(-3) s-1 (where Ki is the dissociation constant of the reversible enzyme-inhibitor complex and k+2 is the rate constant for the inactivation reaction of the enzyme-inhibitor complex)] giving half-lives of inactivation of 30-45 s at saturation. It is argued that the basic-amino-acid residue that abstracts the intramolecularly transferred 4 beta-proton in the reaction mechanism could form a Michael-addition product with compound (I). In contrast, although compound (IV) has a lower inhibition constant (Ki = 14.5 microM), it is a relatively poor alkylating agent (k+2 = 0.13 × 10(-3) s-1). If the conjugated acetylenic ketone groups are replaced by alpha-hydroxyacetylene groups, the resultant analogues of steroids (I)-(IV) are reversible competitive inhibitors with Ki values in the range 27-350 microM. The enzyme binds steroids in the C19 series with functionalized acetylenic substituents at C-17 in preference to steroids in the C18 series bearing similar groups in the ring structure or as C-10 substituents. In the 5,10-seco-steroid series the presence of hydroxy groups at both C-3 and C-17 is deleterious to binding by the enzyme.

scholarly journals Human plasma kallikrein. A rapid purification method with high yield

1981 ◽  
Vol 193 (1) ◽  
pp. 187-192 ◽  
Author(s):  
H Nagase ◽  
A J Barrett
Keyword(s):  
Human Plasma ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Yield ◽  
Gel Chromatography ◽  
Purification Method ◽  
Simple Method ◽  
Bean Trypsin Inhibitor ◽  
Rapid Purification ◽  
Sepharose 4B

A simple method for isolation of kallikrein from human plasma is described. Before activation of the enzyme with acetone, the plasma was treated with 0.2 M-methylamine at pH 8.2 to inactivate alpha 2-macroglobulin and thus prevent the irreversible binding of the active enzyme to the inhibitor. The enzyme was adsorbed on soya-bean trypsin inhibitor-Sepharose 4B and eluted with 5 mM-NaOH, pH 11.3. It was further purified by immunoadsorption of contaminating proteins, and gel chromatography on Ultrogel AcA 44. About 3 mg of kallikrein was obtained from 400 ml of plasma (35% yield). The purified enzyme was shown to be homogeneous by electrophoretic and immunological criteria. The specific activities against benzyloxycarbonylphenylalanylarginine methylcoumarylamide, prolylphenylalanylarginine methylcoumarylamide and tosylarginine methyl ester were higher than any previously reported. The purified enzyme was resolved into two forms of mol.wts. 88 000 and 86 000 in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis without reduction. Each consisted of three chains linked by disulphide bonds, one containing the reactive serine residue (mol.wt. 36 000 or 34 000), and two additional chains (mol.wt. 28 000 and 22 000).

scholarly journals ADP binding to myosin cross-bridges and its effect on the cross-bridge detachment rate constants.

1987 ◽  
Vol 89 (6) ◽  
pp. 905-920 ◽  
Author(s):  
M Schoenberg ◽  
E Eisenberg
Keyword(s):  
Dissociation Constant ◽  
Active Site ◽  
Rate Constants ◽  
Competitive Inhibition ◽  
Psoas Muscle ◽  
Nucleotide Analogues ◽  
Cross Bridge ◽  
Detachment Rate ◽  
The Cross ◽  
Cross Bridges

We have studied the binding of adenosine diphosphate (ADP) to attached cross-bridges in chemically skinned rabbit psoas muscle fibers and the effect of that binding on the cross-bridge detachment rate constants. Cross-bridges with ADP bound to the active site behave very similarly to cross-bridges without any nucleotide at the active site. First, fiber stiffness is the same as in rigor, which presumably implies that, as in rigor, all the cross-bridges are attached. Second, the cross-bridge detachment rate constants in the presence of ADP, measured from the rate of decay of the force induced by a small stretch, are, over a time scale of minutes, similar to those seen in rigor. Because ADP binding to the active site does not cause an increase in the cross-bridge detachment rate constants, whereas binding of nucleotide analogues such as adenyl-5'-yl imidodiphosphate (AMP-PNP) and pyrophosphate (PPi) do, it was possible, by using ADP as a competitive inhibitor of PPi or AMP-PNP, to measure the competitive inhibition constant and thereby the dissociation constant for ADP binding to attached cross-bridges. We found that adding 175 microM ADP to 4 mM PPi or 4 mM AMP-PNP produces as much of a decrease in the apparent cross-bridge detachment rate constants as reducing the analogue concentration from 4 to 1 mM. This suggests that ADP is binding to attached cross-bridges with a dissociation constant of approximately 60 microM. This value is quite similar to that reported for ADP binding to actomyosin subfragment-1 (acto-S1) in solution, which provides further support for the idea that nucleotides and nucleotide analogues seem to bind about as strongly to attached cross-bridges in fibers as to acto-S1 in solution (Johnson, R.E., and P. H. Adams. 1984. FEBS Letters. 174:11-14; Schoenberg, M., and E. Eisenberg. 1985. Biophysical Journal. 48:863-871; Biosca, J.A., L.E. Greene, and E. Eisenberg. 1986. Journal of Biological Chemistry. 261:9793-9800).

scholarly journals Isolation of a high-density-lipoprotein conversion factor from human plasma. A possible role of apolipoprotein A-IV as its activator

1988 ◽  
Vol 254 (1) ◽  
pp. 179-184 ◽  
Author(s):  
P J Barter ◽  
O V Rajaram ◽  
L B F Chang ◽  
K A Rye ◽  
P Gambert ◽  
...  
Keyword(s):  
High Density Lipoprotein ◽  
Human Plasma ◽  
Conversion Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Density Lipoprotein ◽  
Exchange Chromatography ◽  
High Density ◽  
Major Band ◽  
Homogeneous Population ◽  
Apolipoprotein A

1. A high-density-lipoprotein (HDL) conversion factor was partially purified from human plasma by precipitation with (NH4)2SO4, ultracentrifugation, cation-exchange chromatography, anion-exchange chromatography and chromatography on a column of hydroxyapatite. 2. This factor modulates the particle size of HDL by converting a homogeneous population into new populations of particles, some of which are smaller and others larger than those in the original population. 3. The isolated HDL conversion factor appeared as one major band and at least three minor bands on SDS/polyacrylamide-gel electrophoresis; attempts to purify this factor further resulted in loss of conversion activity. 4. Preparations of the HDL conversion factor were stable after heating to 58 degrees C for 1 h, and were shown not to possess proteolytic activity. 5. The conversion factor was distinct from the known apolipoproteins, none of which had HDL conversion activity. 6. Addition of apolipoprotein A-IV had a dose-dependent potentiating effect on the process promoted by the HDL conversion factor.

scholarly journals A 1,4-β-glucan glucanohydrolase from the cellulolytic fungus Trichoderma viride QM 9414. Purification, characterization and preparation of an immunoadsorbent for the enzyme

1979 ◽  
Vol 179 (1) ◽  
pp. 141-149 ◽  
Author(s):  
U Håkansson ◽  
L G Fägerstam ◽  
L G Pettersson ◽  
L Andersson
Keyword(s):  
Isoelectric Focusing ◽  
Molecular Sieve ◽  
Polyacrylamide Gel Electrophoresis ◽  
Trichoderma Viride ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Beta Glucan ◽  
Ph Values ◽  
Sepharose 4B

A 1,4-beta-glucan glucanohydrolase (EC 3.2.1.4) was isolated from culture filtrates of the fungus Trichoderma viride QM 9414 by molecular-sieve chromatography on Bio-Gel P-30, ion-exchange chromatography on DEAE-Sephadex A-50 and isoelectric focusing in a density gradient. Polyacrylamide-gel electrophoresis at two different pH values, analytical isoelectric focusing in a polyacrylamide-gel slab and molecular-sieve chromatography of the reduced and alkylated enzyme in a denaturing medium indicated a homogeneous protein. The enzyme has a mol.wt. of 51,000 and is not a glycoprotein. The pI was found to be 4.66 at 23 degrees C. Antiserum against the purified enzyme was prepared and the amount of enzyme in the original filtrate was determined by rocket immunoelectrophoresis to be about 50mg/liter. An immunoadsorbent made from CNBr-activated sepharose 4B and antiserum affords a rapid and highly specific purification of the enzyme.

Purification of human plasma lecithin:cholesterol acyltransferase and its activation by metal ions

1980 ◽  
Vol 58 (7) ◽  
pp. 539-541 ◽  
Author(s):  
Ginzaburo Suzue ◽  
Camilla Vézina ◽  
Yves L. Marcel
Keyword(s):  
Human Plasma ◽  
Gel Electrophoresis ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Lecithin:Cholesterol Acyltransferase ◽  
Preparative Electrophoresis ◽  
Preparative Ultracentrifugation ◽  
Apo Ai

Lecithin:cholesterol acyltransferase (LCAT) has been purified from human plasma by sequential preparative ultracentrifugation, ion-exchange chromatography on DEAE-Sephacel, and affinity chromatography on HDL-Sepharose and on wheat germ agglutinin-Sepharose. After the final step, which included preparative electrophoresis or alternatively, chromatography on hydroxylapatite, a purification of about 24 000-fold was obtained. The LCAT preparation was pure according to alkaline polyacrylamide and SDS–polyacrylamide gel electrophoresis and did not react against antisera to apo AI, AII, and D. The LCAT preparation obtained by preparative electrophoresis was stimulated by Cu2+, Ni2+, Co2+, and Zn2+ at both stages of the reaction, phospholipase reaction and cholesterol esterification. This stimulatory effect was abolished by EDTA.

scholarly journals Mevalonate kinase in green leaves and etiolated cotyledons of the French bean Phaseolus vulgaris

1973 ◽  
Vol 133 (2) ◽  
pp. 335-347 ◽  
Author(s):  
J. C. Gray ◽  
R. G. O. Kekwick
Keyword(s):  
Phaseolus Vulgaris ◽  
Competitive Inhibition ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
French Bean ◽  
Exchange Chromatography ◽  
Mevalonate Kinase ◽  
Ph Optimum ◽  
Enzyme Preparations ◽  
Green Leaves

1. Partially purified preparations of mevalonate kinase were obtained from green leaves and etiolated cotyledons of Phaseolus vulgaris. 2. After removal of interfering polyphenols both enzyme preparations behaved identically on gel filtration, ion-exchange chromatography and density-gradient centrifugation. 3. The kinetic parameters of the preparations from the two sources were indistinguishable. The preparation from etiolated cotyledons had a Km of 4.26×10−5m for RS-mevalonate and 1.54×10−3m for ATP. The preparation from green leaves had a Km of 4.55×10−5m for RS-mevalonate and 1.75×10−3m for ATP. The pH optimum of both enzyme preparations was pH7.0. 4. The effect of inhibitors on the two enzyme preparations was similar, both being inhibited by reagents known to react with thiol groups, and the two preparations had similar inhibitor constants for competitive inhibition by prenyl pyrophosphates. 5. The molecular weight of the enzyme in both preparations was estimated to be 100000; the enzymes from the two preparations had similar mobilities on polyacrylamide-gel electrophoresis.

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