scholarly journals Studies on the temperature-dependent autoinhibition of human plasma kallikrein I

1982 ◽  
Vol 205 (3) ◽  
pp. 529-534 ◽  
Author(s):  
P R Levison ◽  
G Tomalin
Keyword(s):  
Human Plasma ◽  
Initial Velocity ◽  
Sodium Deoxycholate ◽  
Enzyme Concentration ◽  
Low Molecular Weight ◽  
Plasma Kallikrein ◽  
Kinetic Effect ◽  
Temperature Dependent ◽  
Endogenous Inhibitors ◽  
Hydrolysis Of

At 37 degrees C, human plasma kallikrein I follows Michaelis-Menten behaviour and exhibits a normal linear relationship between the initial velocity of hydrolysis of Ac-Pro-Phe-Arg-OMe, HCl and enzyme concentration in the range 0-150 pM. At temperatures of 30 degrees C and below substantial deviations from linearity are observed over the same enzyme concentration range. The temperature-dependent autoinhibition of kallikrein I activity is reversible and is not due to low-molecular-weight endogenous inhibitors or cofactors. The kinetic effect is apparently due to aggregation and can be abolished by the addition of sodium deoxycholate.

scholarly journals The kinetics of hydrolysis of some extended N-aminoacyl-l-arginine methyl esters by human plasma kallikrein. Evidence for subsites S2 and S3

1982 ◽  
Vol 203 (1) ◽  
pp. 149-153 ◽  
Author(s):  
P R Levison ◽  
G Tomalin
Keyword(s):  
Human Plasma ◽  
Methyl Esters ◽  
Plasma Kallikrein ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Rate Limiting Step ◽  
Kinetics Of Hydrolysis ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Hydrolysis Of

Subsites in the S2-S4 region were identified in human plasma kallikrein. Kinetic constants (kcat., Km) were determined for a series of seven extended N-aminoacyl-L-arginine methyl esters based on the C-terminal sequence of bradykinin (-Pro-Phe-Arg) or (Gly)n-Arg. The rate-limiting step for the enzyme-catalysed reaction was found to be deacylation of the enzyme. It was possible to infer that hydrogen-bonded interactions occur between substrate and the S2-S4 region of kallikrein. Insertion of L-phenylalanine at residue P2 demonstrates that there is also a hydrophobic interaction with subsite S2, which stabilizes the enzyme-substrate complex. The strong interaction demonstrated between L-proline at residue P3 and subsite S3 is of greatest importance in the selectivity of human plasma kallikrein. The purification of kallikrein from Cohn fraction IV of human plasma is described making use of endogenous Factor XIIf to activate the prekallikrein. Kallikreins I (Mr 91 000) and II (Mr 85 000) were purified 170- and 110-fold respectively. Kallikrein I was used for the kinetic work.

Human plasma kallikrein. Preliminary studies on hydrolysis of proteins and peptides

1978 ◽  
Vol 8 (1-2) ◽  
pp. 125-131 ◽  
Author(s):  
Claudio A. M. Sampaio ◽  
Daisy Grisolia
Keyword(s):  
Human Plasma ◽  
Plasma Kallikrein ◽  
Hydrolysis Of ◽  
Proteins And Peptides

S1′ and S2′ subsite specificities of human plasma kallikrein and tissue kallikrein 1 for the hydrolysis of peptides derived from the bradykinin domain of human kininogen

2008 ◽  
Vol 389 (12) ◽  
Author(s):  
Aurelio Resende Lima ◽  
Fabiana M. Alves ◽  
Pedro Francisco Ângelo ◽  
Douglas Andrade ◽  
Sachiko I. Blaber ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Plasma ◽  
Receptor Agonist ◽  
Plasma Kallikrein ◽  
Tissue Kallikrein ◽  
Charged Amino Acids ◽  
B1 Receptor ◽  
Lower Specificity ◽  
Natural Amino Acids ◽  
Hydrolysis Of

AbstractThe S1′ and S2′ subsite specificities of human tissue kallikrein 1 (KLK1) and human plasma kallikrein (HPK) were examined with the peptide series Abz-GFSPFRXSRIQ-EDDnp and Abz-GFSPFRSXRIQ-EDDnp [X=natural amino acids or S(PO3H2)]. KLK1 efficiently hydrolyzed most of the peptides except those containing negatively charged amino acids at P1′ and P2′ positions. Abz-GFSPFRSSRIQ-EDDnp, as in human kininogen, is the best substrate for KLK1 and exclusively cleaved the R-S bond. All other peptides were cleaved also at the F-R bond. The synthetic human kininogen segment Abz-MISLMKRPPGFSPFRS390S391RI-NH2was hydrolyzed by KLK1 first at R-S and then at M-K bonds, releasing Lys-bradykinin. In the S390and S391phosphorylated analogs, this order of hydrolysis was inverted due to the higher resistance of the R-S bond. Abz-MISLMKRPPG-FSPFRSS(PO3H2)391RI-NH2was hydrolyzed by KLK1 at M-K and mainly at the F-R bond, releasing des-(Arg9)-Lys-Bk which is a B1 receptor agonist. HPK cleaved all the peptides at R and showed restricted specificity for S in the S1′ subsite, with lower specificity for the S2′ subsite. Abz-MISLMKRPPGFSPFRSSRI-NH2was efficiently hydrolyzed by HPK under bradykinin release, while the analogs containing S(PO3H2) were poorly hydrolyzed. In conclusion, S1′ and S2′ subsite specificities of KLK1 and HPK showed peculiarities that were observed with substrates containing the amino acid sequence of human kininogen.

scholarly journals The kinetics of hydrolysis of some extended N-aminoacyl-l-arginine methyl esters by porcine pancreatic kallikrein. A comparison with human plasma Kallikrein

1982 ◽  
Vol 203 (1) ◽  
pp. 299-302 ◽  
Author(s):  
P R Levison ◽  
G Tomalin
Keyword(s):  
Human Plasma ◽  
Methyl Esters ◽  
Catalytic Efficiency ◽  
Plasma Kallikrein ◽  
Kinetics Of Hydrolysis ◽  
Relative Differences ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Limiting Values

The effects of subsite interactions in the S2-S4 region [Schechter & Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162] of porcine pancreatic kallikrein (EC 3.4.21.8) on its catalytic efficiency have been investigated. Kinetic constants (Kcat, Km) have been determined for a series of seven extended N-aminoacyl-L-arginine methyl esters whose sequence is based on either the C-terminal sequence of kallidin (-Pro-Phe-Arg) or (-Gly-)nArg. With these substrates it has been found that neither acylation nor deacylation of the enzyme is rate-limiting. Values of Kcat. range from 21.5 to 2320s-1, indicating that there are interactions with different residues in the N-aminoacyl chain and enzyme subsites in the S2-S4 region. It is shown that possible hydrogen-bonded interactions with the enzyme in the S3-S4 region have a significant effect on catalysis. The presence of L-phenylalanine at P2 has a very large effect on both Kcat, and Km, giving a greatly enhanced catalytic efficiency. Substrates with L-proline at P3 also have a marked effect, but in this case the overall effect is one of lowered catalytic efficiency. By comparison with the results of a similar study with human plasma kallikrein I (EC 3.4.21.8), it has been possible to demonstrate that there are considerable differences in kinetic behaviour between the two enzymes. These are related to relative differences in the rates of acylation and deacylation with ester substrates and also the roles of subsites S2 and S3 of the two enzymes.

Chromogenic Substrates And Activated Forms Of FXII: Comparison Between The α- And β-Forms And Selectivity With Regard To Plasma Kallikrein And FXIa

1981 ◽  
Author(s):  
L Aurell ◽  
S Gustavsson ◽  
P Friberger
Keyword(s):  
Enzyme Concentration ◽  
Plasma Kallikrein ◽  
Small Contribution ◽  
Plasma Samples ◽  
Contact Activation ◽  
Chromogenic Substrates ◽  
Measuring Range ◽  
Specific Activities ◽  
Hydrolysis Of ◽  
Plasma Assay

Activated FXII of both the α-(MW 80000) and the β-(MW 28000) form was tested with a large number of chromogenic tri- and tetrapeptides. The activities of the two enzymes correlated well (r=0.84, n=30). The α-form appeared to have a somewhat higher specificity.The plasma kallikrein substrate S-2302 and the FXa substrate S-2222 have earlier been shown to be the commercially available substrates most sensitive to the β-form of activated FXII . The specific activities of both the α-and β-forms are the same with S-2302 in the system tris buffer pH 8.0 and I 0.05 at 37°C. At the enzyme concentration of 4 nmol/1, the change in absorbance per min was 0.02 which is in the suitable measuring range. This concentration is approximately equivalent to fully activated plasma diluted 1:100.From these findings, it could be confirmed that when plasma prekallikrein is assayed via contact activation with S-2302, there is a definite but small contribution to the hydrolysis of the substrate from activated FXII.A more selective substrate for activated FXII with regard to plasma kallikrein would be preferable in a plasma assay of FXII. S-2222 has these characteristics. At the substrate concentration of 0.33 mmol/1, the β-form of activated FXII splits S-2222 essentially as efficiently as S-2302 while plasma kallikrein is only 5% as efficient with S-2222 as with S-2302.Both types of substrates are insensitive to FXIa at concentrations obtainable in plasma samples suitably diluted for the assaying of FXII and plasma prekallikrein.

On the Preparation of Bovine α-Thrombin

1978 ◽  
Vol 39 (01) ◽  
pp. 193-200 ◽  
Author(s):  
Erwin F Workman ◽  
Roger L Lundblad
Keyword(s):  
Immobilized Enzyme ◽  
Catalytic Properties ◽  
Specific Activity ◽  
Guanidine Hydrochloride ◽  
Low Molecular Weight ◽  
Reversible Process ◽  
Gel Beads ◽  
Tissue Thromboplastin ◽  
C 50 ◽  
Hydrolysis Of

SummaryAn improved method for the preparation of bovine α-thrombin is described. The procedure involves the activation of partially purified prothrombin with tissue thromboplastin followed by chromatography on Sulfopropyl-Sephadex C-50. The purified enzyme is homogeneous on polyacrylamide discontinuous gel electrophoresis and has a specific activity toward fibrinogen of 2,200–2,700 N.I.H. U/mg. Its stability on storage in liquid media is dependent on both ionic strenght and temperature. Increasing ionic strength and decreasing temperature result in optimal stability. The denaturation of α-thrombin by guanidine hydrochloride was found to be a partially reversible process with the renatured species possessing properties similar to “aged” thrombin. In addition, the catalytic properties of a-thrombin covalently attached to agarose gel beads were also examined. The activity of the immobilized enzyme toward fibrinogen was affected to a much greater extent than was the hydrolysis of low molecular weight, synthetic substrates.

Studies on Activator Formation in Human Plasma with Streptokinase

1973 ◽  
Vol 30 (02) ◽  
pp. 381-392
Author(s):  
M Martin ◽  
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Mass Action ◽  
Activator Concentration ◽  
Kinetic Effect ◽  
Mass Action Law

SummaryThe plasminogen-streptokinase complex called “activator” was present in diluted plasma in the form of a largely dissociated mixture. More than ⅞ of the streptokinase and plasminogen molecules were available for further activator formation.The activator is probably a dissociated complex of the formulaStreptokinase + Plasminogen ⇄ Activator.The fact that an increase in activator concentration by x times is obtained by multiplying either the streptokinase content by the factor y or the plasminogen concentration by the same factor y would point to a kinetic effect along the lines of the mass action law.

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