A New Method for Plasminogen Standardization

SummaryA method for the standardization of human plasminogen is proposed, based on the stoichiometric interaction between plasminogen and streptokinase, resulting in inhibition of proteolytic activity. Activation of a constant amount of plasminogen with increasing amounts of streptokinase yields linearly decreasing activities, as a function of streptokinase, with a sharp transition to a constant residual level. The point of transition corresponds to complete saturation of plasmin with streptokinase in a 1:1 molar ratio, and is therefore a measure of the amount of plasminogen present initially, in terms of streptokinase equivalents. The equivalence point is independent of the kind of protein substrate used, buffer, pH, length of digestion and, within limits, temperature. The method, therefore, is not subject to the variations commonly encountered in the usual determination based on specific activity measurements.

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Mechanism of Human Plasminogen Activation by Streptokinase and Human Plasmin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654808 ◽

1964 ◽

Vol 11 (01) ◽

pp. 085-093

Author(s):

W. F Blatt ◽

JL Gray ◽

H Jensen

Keyword(s):

Proteolytic Activity ◽

Low Ph ◽

Plasminogen Activation ◽

Sensitive Tool ◽

Human Plasminogen

SummaryA sensitive tool has been described for measuring fibrinolysis in reconstituted systems using thrombelastography. Activator mixtures with no appreciable proteolytic activity can similarly be tested in this system when the fibrinogen utilized has sufficient plasminogen present. Exposure of human plasminstreptokinase mixtures formed at pH 7.0 to acid conditions produced a striking loss of activator activity which could not be ascribed to low pH lability of the components, nor to plasmin action on the SK at pH 2.0. This is additional evidence for the hypothesis that human plasmin interacts with SK to form a complex capable of converting human and bovine plasminogen to plasmin.

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The nine amino-terminal residues of delta-aminolevulinate synthase direct beta-galactosidase into the mitochondrial matrix

Molecular and Cellular Biology ◽

10.1128/mcb.6.2.355-364.1986 ◽

1986 ◽

Vol 6 (2) ◽

pp. 355-364

Author(s):

T Keng ◽

E Alani ◽

L Guarente

Keyword(s):

Amino Acid ◽

Fusion Proteins ◽

Specific Activity ◽

Nuclear Gene ◽

Yeast Cells ◽

Mitochondrial Matrix ◽

Aminolevulinate Synthase ◽

Amino Terminal ◽

Activity Measurements ◽

Beta Galactosidase

delta-Aminolevulinate synthase, the first enzyme in the heme biosynthetic pathway, is encoded by the nuclear gene HEM1. The enzyme is synthesized as a precursor in the cytoplasm and imported into the matrix of the mitochondria, where it is processed to its mature form. Fusions of beta-galactosidase to various lengths of amino-terminal fragments of delta-aminolevulinate synthase were constructed and transformed into yeast cells. The subcellular location of the fusion proteins was determined by organelle fractionation. Fusion proteins were found to be associated with the mitochondria. Protease protection experiments involving the use of intact mitochondria or mitoplasts localized the fusion proteins to the mitochondrial matrix. This observation was confirmed by fractionation of the mitochondrial compartments and specific activity measurements of beta-galactosidase activity. The shortest fusion protein contains nine amino acid residues of delta-aminolevulinate synthase, indicating that nine amino-terminal residues are sufficient to localize beta-galactosidase to the mitochondrial matrix. The amino acid sequence deduced from the DNA sequence of HEM1 showed that the amino-terminal region of delta-aminolevulinate synthase was largely hydrophobic, with a few basic residues interspersed.

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SELECTED PROTEOLYTIC ACTIVITY BY EXTRACTS OF DERMESTID LARVAE

Southern Brazilian Journal of Chemistry - Southern Brazilian Journal of Chemistry, Volume 28, No. 28, 2020 ◽

10.48141/sbjchem.v19.n19.2011.82_2011.pdf ◽

2011 ◽

Vol 19 (19) ◽

pp. 79-83

Author(s):

Lavinel G. IONESCU

Keyword(s):

Enzyme Activity ◽

Ammonium Sulfate ◽

Proteolytic Activity ◽

Specific Activity ◽

Proteolytic Enzymes ◽

High Specific Activity ◽

Water Extract ◽

Dermestes Maculatus ◽

Crude Preparation

The larvae of the Beetle Dermestes maculatus De Geer can subsist on a diet consisting largely of protein. Studies have been undertaken to investigate the nature of proteolytic enzymes. A water extract of the larvae yielded a crude preparation that hydrolyzes gelatin, bide powder, hemoglobin substrate, benzoyl-DL-arginine p-nitroamilide, and glutaryl-L-phenylalanine p-nitroanilide. Enzyme activity was found in a non-dialyzable, heat- and acid0labile portion of the extract yielded two fractions with high specific activity towards gelatin. These are precipitated between 40% to 60% saturation of ammonium sulfate and 60% to 80% saturation. The higher specific activity was observed in the 40%-60% fraction. These results suggest that the larvae of these dermestids contain proteolytic enzymes with actions similar to mammalian trypsin and chymotrypsin. The results also suggest that other proteolytic enzymes may be present as well.

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Silver Cation Coordination Study to AsW9 Ligand – A Trilacunar Arsenotungstate Compound

Acta Medica Marisiensis ◽

10.1515/amma-2017-0018 ◽

2017 ◽

Vol 63 (2) ◽

pp. 70-72

Author(s):

Lavinia Berta ◽

Andrei Gâz ◽

Francisc Boda ◽

Augustin Curticapean

Keyword(s):

Graphical Method ◽

Ph Value ◽

Building Blocks ◽

Silver Cation ◽

Molar Ratio ◽

Equivalence Point ◽

Tetrahedral Geometry ◽

Sandwich Type ◽

Preliminary Study ◽

Cation Coordination

Abstract Objective: The main objective of this research is to find the coordination ratio between AsW9 and Ag+, as a preliminary study for synthesizing a new silver-arsenotungstate complex. Material and method: The ligand:cation molar ratio in complexes was determined by conductometric and potentiometric titrations of AsW9 with silver salts: CH3COOAg, AgNO3. Results: The ratio was obtained from the inflexion points of the curves when molar ratio was plotted versus conductivity, or from the equivalence point when silver added volume was plotted versus pH value. Each graphic shows one point of inflexion corresponding to 1:1.54 ratio of AsW9:Ag+. In the same manner, the equivalent volumes determined by graphical method gave the ratio 1:1.53. The spectral results confirmed that a AsW9:Ag+ complex was formed since the ligand absorption maxima values have been changed from 190 nm to 197 nm in the case of using AgNO3 and 196 nm for CH3COOAg corresponding to the W=Od bond, and from 246.5 nm to 274 nm (AgNO3) and 270 nm (CH3COO-Ag+) for the W-Ob,c-W bond. Conclusions: Silver cation exhibit a preference for AsW9 in a ratio of 3 to 2. This ratio can be associated to a sandwich type arrangement, with two trilacunary Keggin building blocks incorporating 3 metal cations in a tetrahedral geometry.

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Effects of vitamin D-induced chronic hypercalcemia on rat renal cortical plasma membranes and mitochondria

AJP Renal Physiology ◽

10.1152/ajprenal.1987.252.2.f267 ◽

1987 ◽

Vol 252 (2) ◽

pp. F267-F275 ◽

Cited By ~ 5

Author(s):

M. Levi ◽

B. A. Molitoris ◽

T. J. Burke ◽

R. W. Schrier ◽

F. R. Simon

Keyword(s):

Brush Border ◽

Cell Injury ◽

Specific Activity ◽

Total Phospholipid ◽

Basolateral Membrane ◽

Calcium Content ◽

Molar Ratio ◽

Renal Tubular Cell ◽

Molar Composition ◽

Renal Tubular

Increases in intracellular and mitochondrial calcium content that accompany ischemic and toxic acute renal failure have been suggested to mediate renal tubular cell injury and dysfunction, but the mechanism(s) are unknown. We studied the effects of in vivo vitamin D-induced chronic hypercalcemia on rat renal cortical brush-border and basolateral membranes and mitochondria. In the brush-border membrane, hypercalcemia caused significant decreases in alkaline phosphatase-specific activity, total phospholipid molar content, and phosphatidylserine percent molar composition and increases in the cholesterol-to-total phospholipid molar ratio and phosphatidylinositol percent molar composition. In the basolateral membrane, hypercalcemia caused significant decreases in Na+-K+-ATPase-specific activity and total phospholipid molar content and increases in the cholesterol-to-total phospholipid molar ratio and phosphatidylinositol 4,5-bisphosphate percent molar composition. In the mitochondria, hypercalcemia caused a mild increase in the mitochondrial calcium content, but no alterations in succinic dehydrogenase-specific activity, succinate-, ADP-, or uncoupler-induced respiration. Thus hypercalcemia caused alterations in brush-border and basolateral membrane enzyme activity and lipid composition, but no functional changes were detected in mitochondria. These hypercalcemia-induced plasma membrane biochemical alterations may be markers of early cell injury and suggest a role for calcium in causing or predisposing to renal tubular cell injury.

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Calcium Determinations in Kinetic Studies

Clinical Chemistry ◽

10.1093/clinchem/13.9.760 ◽

1967 ◽

Vol 13 (9) ◽

pp. 760-768 ◽

Cited By ~ 4

Author(s):

Joan Harrison ◽

A J W Hitchman ◽

J M Finlay

Keyword(s):

Specific Activity ◽

Kinetic Studies ◽

Accurate Method ◽

Urine Calcium ◽

Blood Calcium ◽

Exchangeable Calcium ◽

Published Evidence ◽

Activity Measurements

Abstract Published reports of a fraction of blood calcium that does not equilibrate with a tracer has created a controversy that challenges the validity of much calcium kinetic data. To resolve this controversy, calcium specific-activity measurements were made on blood and urine samples in vitro and in vivo. The experiments were designed to provide maximal sensitivity for demonstrating non exchangeable calcium. A new and accurate method for urine calcium determinations was used. The results demonstrated complete equilibration between tracer and stable blood calcium. We suggest that published evidence of non exchangeable calcium in blood and urine is erroneous due to inaccurate urine calcium determinations.

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The unactivated form of the first component of human complement, C1

Biochemical Journal ◽

10.1042/bj1570541 ◽

1976 ◽

Vol 157 (3) ◽

pp. 541-548 ◽

Cited By ~ 122

Author(s):

I Gigli ◽

R R Porter ◽

R B Sim

Keyword(s):

Human Serum ◽

Proteolytic Activity ◽

Molar Ratio ◽

Human Complement ◽

Peptide Bonds ◽

Optimum Activity ◽

Haemolytic Assay ◽

Subsequent Loss

The first component of complement, C1, was isolated unactivated from human serum by repeated additions of di-isopropyl phosphorofluoridate during isolation. The unactivated subcomponents were also isolated, and evidence is given that the three subcomponents C1q, C1r and C1s account wholly for the activity of component C1 in serum. No evidence could be found for a fourth subcomponent, C1t. The approximate molar proportions of the subcomponents in serum are C1q/C1r/C1s = 1:2:2. Optimum activity by haemolytic assay was found at approximate molar proportions C1q/C1r/C1s of 1:4:4. No activity was found when subcomponents were assayed singly or in pairs, except for subcomponents C1q and C1s, which in molar ratio 1:4 gave 15-20% of the activity of the mixture C1q + C1r + C1s. The proteolytic activity of the isolated subcomponent C1s varied according to the method of activation used. Subcomponents C1q + C1r + C1s and C1q + C1s in the presence of antibody-antigen aggregates were activated and inactivated simultaneously, showing a peak of activity and subsequent loss of activity. Both reactions are probably due to proteolysis, and analysis of the peptide bonds split will be necessary to distinguish these two phenomena.

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Effect of PAI-1 levels on the molar concentrations of active tissue plasminogen activator (t-PA) and t-PA/PAI-1 complex in plasma

Blood ◽

10.1182/blood.v76.5.930.bloodjournal765930 ◽

1990 ◽

Vol 76 (5) ◽

pp. 930-937 ◽

Cited By ~ 1

Author(s):

WL Chandler ◽

SL Trimble ◽

SC Loo ◽

D Mornin

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Plasminogen Activator Inhibitor ◽

Molar Ratio ◽

Molar Activity ◽

Activity Measurements ◽

Tissue Plasminogen ◽

Activator Inhibitor Type ◽

Pai 1

We determined the in vivo molar concentrations of active tissue plasminogen activator (t-PA), active plasminogen activator inhibitor type 1 (PAI-1), and t-PA/PAI-1 complex. t-PA activity was measured in plasma stabilized by immediate acidification. PAI-1 activity and t- PA/PAI-1 complex antigen were measured in citrated plasma; these measurements were corrected for the loss in PAI-1 activity and increase in complex that occurs in unacidified plasma samples due to the continued reaction between t-PA and PAI-1 after the sample was drawn. To convert t-PA and PAI-1 activity measurements into molar concentrations we determined the specific molar activity of t-PA and PAI-1 in vivo: 4.48 x 10(13) IU/mol. Of 72 subjects studied, 13 had less than 150 pmol/L active PAI-1; in these individuals 33% +/- 21% of their t-PA was active and the molar ratio of active t-PA to active PAI- 1 was 0.20 +/- 0.13. In the 11 subjects with greater than 500 pmol/L active PAI-1, 1.5% = 1.1% of the t-PA was active and the molar ratio of active t-PA to active PAI-1 was 0.0043 +/- 0.0036. Overall, the fraction of active t-PA declined exponentially as a function of the active PAI-1 concentration. During the day, the percentage of total t- PA that was active increased from 12% at 8:00 AM to 31% at 8:00 PM, while the molar ratio of active t-PA to active PAI-1 increased from 0.05 to 0.22 from morning to evening (n = 12).

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