Limited Proteolysis of the Purified Aα Chain of Human Fibrinosen by Plasmin

1975 ◽  
Author(s):  
L. Williams ◽  
G. Murano
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Peptide Mapping ◽  
Proteolytic Enzymes ◽  
Degradation Products ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Terminal Region ◽  
Low Molecular Weight ◽  
Human Fibrinogen

Based on evidence that a portion of circulating fibrinogen consists of a family of catabolic intermediates formed by proteolytic degradation of the COOH terminal region of Aα chains, we attempted to obtain early degradation products using the purified alkylated Aα chain derivative of human fibrinogen as the substrate and plasmin as the enzyme. Having established optimal conditions, a preparative quantity of material was digested in 0.1 M tris buffer pH = 9.5; time = 4 min; E/S ratio = 1/75 (mole/mole); temp = 37° C. Low molecular weight fragments were separated from the larger species, and further purified by gel filtration on Sephadex G-100. Selected early fragments were analyzed by polycrylamide gel electrophoresis, amino acid composition, peptide mapping and partial N-terminal amino acid sequence. Two of the earliest low molecular weight fragments released by plasmin were derived from the N-terminal region of the Aα chain. Their molecular size was estimated at about 10,000 daltons. One fragment contains fibrinopeptide A; both fragments extend beyond Met-51. Our data indicate that: a) the specificity of plasmin on the purified Aα chain differs from that on intact fibrinogen; or b) proteolytic enzymes other than or in addition to plasmin are responsible for the formation of early catabolic fibrinogen intermediates having a degraded Aα chain.(Supported by USPHS N. I. H. Grant HL 14142.)

scholarly journals Evidence that functional subunits of antihemophilic factor (Factor VIII) are linked by noncovalent bonds

Blood ◽  
1976 ◽  
Vol 48 (1) ◽  
pp. 87-94 ◽  
Author(s):  
MC Poon ◽  
OD Ratnoff
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Protease Inhibitors ◽  
Proteolytic Enzymes ◽  
Gel Filtration ◽  
Factor Ix ◽  
Low Molecular Weight ◽  
Native State ◽  
Noncovalent Bonds ◽  
Antihemophilic Factor

Abstract Partially purified human antihemophilic factor (AHF, factor VIII), when treated with high concentrations of salt, has been shown to dissociate into two components: one, of relatively low molecular weight, possesses procoagulant activity, and the other, of higher molecular weight, forms precipitates with heterologous antiserum against AHF and supports ristocetin-induced platelet aggregation. The ease of separation suggests that the two components in the native state might be held together by noncovalent bonds. Earlier observations do not exclude the possibility that the subunits may be covalently bonded in nature but might be severed by plasma proteolytic enzymes during laboratory manipulation. The issue was examined by preparing partially purified AHF from fresh human plasma in the presence of protease inhibitors, including benzamidine, soybean trypsin inhibitor, epsilon-aminocaproic acid, heparin, and hirudin. Under these conditons, gel filtration in the presence of 0.25 M calcium chloride and 0.001 M benzamidine resulted in its separation into two components, having properties identical to those separated in the absence of these protease inhibitors. The inhibitor mixture blocked generation and action of streptokinase- and kaolin-activated plasmin from plasma, and protected both plasma AHF and partially purified AHF from the action of thrombin. Surface-induced activation of PTA (factor XI) was partially inhibited, and that of Christmas factor (factor IX) was completely inhibited. This observation provides further evidence that in the native state the high- and low-molecular-weight components of preparations of antihemophilic factor are held together by noncovalent bonds.

scholarly journals A Low Molecular Weight Fibrinolytic Enzyme from Polymorphonuclear Leukocytes (PMN)

1977 ◽  
Author(s):  
L.A. Moroz
Keyword(s):  
Molecular Weight ◽  
Solid Phase ◽  
Degradation Products ◽  
Gel Filtration ◽  
Protein Band ◽  
Fibrinolytic Enzyme ◽  
Low Molecular Weight ◽  
Cationic Protein ◽  
Alkaline Proteinase ◽  
Solid Phase Assay

Despite evidence implicating PMN in fibrinolysis, the enzymes involved are incompletely characterized.PMN were prepared from normal blood by dextran sedimentation and fibrinolytic activity assayed by l25I-fibrin solid phase assay (Blood 46:543, 1975). More than 80% of activity was associated with intact PMN, was stimulated by Na salicylate (+65%, 20 mg/100 ml) and inhibited by α1-anti-trypsin (α1AT, -48%, 2 × 10-6 M). Similar activity was found in a PMN membrane fraction prepared by homogenization, differential centrifugation and Sepharose 4B gel filtration, from which fraction it was released by freeze-thawing and/or IM KCl treatment. Soluble enzyme activity was inhibited by of α1AT (-80%, 10-6 M), PMSF (-98%, 10-3 M), FeCl3 and ZnCl3 (-100%, 10-2 M), vitamin E(-38%, 10-4 M) and trypan blue (-40%, 10-3 M), but not by EACA (10-2 M), tranexamic acid (10-2 M), TLCK (10-3 M) or TPCK (10-3 M). This activity had an alkaline proteinase pH-activity profile, was localized to a single cationic protein band on acid Polyacrylamide disc gel electrophoresis, with pl of 8.6-8.7 by isoelectric focussing, and eluted between lysozyme and myoglobin on Bio-Gel P-10. On Bio-Gel A 0.5m and P-10, 125I-fibrin degradation products eluted after myoglobin. These findings indicate the presence in PMN of a low molecular weight, membrane-associated fibrinolytic enzyme of alkaline proteinase and serine active site type.

A Simple and Practical Method for Isolation of an Early Plasmin Degradation Product of Human Fibrinogen

1977 ◽  
Vol 37 (03) ◽  
pp. 464-470 ◽  
Author(s):  
Kimiteru Takagi ◽  
Tadashi Kawai
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Degradation Product ◽  
Degradation Products ◽  
Practical Method ◽  
Human Fibrinogen ◽  
Simple Method ◽  
Hydrophobic Amino Acids ◽  
Very High

SummaryOne of the earliest plasmin degradation products of human fibrinogen, so-called fragment A, was isolated by a simple method.This peptide has a molecular weight of approximately 22,500, migrating electrophoretically at beta-area, and its amino acid composition shows a very high content of glycine, serine, threonine and proline, and a markedly low content of hydrophobic amino acids. This fragment does not react against anti-fibrinogen; however, the anti-serum of this fragment reacts strongly with fibrinogen.

scholarly journals Evidence that functional subunits of antihemophilic factor (Factor VIII) are linked by noncovalent bonds

Blood ◽  
1976 ◽  
Vol 48 (1) ◽  
pp. 87-94
Author(s):  
MC Poon ◽  
OD Ratnoff
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Protease Inhibitors ◽  
Proteolytic Enzymes ◽  
Gel Filtration ◽  
Factor Ix ◽  
Low Molecular Weight ◽  
Native State ◽  
Noncovalent Bonds ◽  
Antihemophilic Factor

Partially purified human antihemophilic factor (AHF, factor VIII), when treated with high concentrations of salt, has been shown to dissociate into two components: one, of relatively low molecular weight, possesses procoagulant activity, and the other, of higher molecular weight, forms precipitates with heterologous antiserum against AHF and supports ristocetin-induced platelet aggregation. The ease of separation suggests that the two components in the native state might be held together by noncovalent bonds. Earlier observations do not exclude the possibility that the subunits may be covalently bonded in nature but might be severed by plasma proteolytic enzymes during laboratory manipulation. The issue was examined by preparing partially purified AHF from fresh human plasma in the presence of protease inhibitors, including benzamidine, soybean trypsin inhibitor, epsilon-aminocaproic acid, heparin, and hirudin. Under these conditons, gel filtration in the presence of 0.25 M calcium chloride and 0.001 M benzamidine resulted in its separation into two components, having properties identical to those separated in the absence of these protease inhibitors. The inhibitor mixture blocked generation and action of streptokinase- and kaolin-activated plasmin from plasma, and protected both plasma AHF and partially purified AHF from the action of thrombin. Surface-induced activation of PTA (factor XI) was partially inhibited, and that of Christmas factor (factor IX) was completely inhibited. This observation provides further evidence that in the native state the high- and low-molecular-weight components of preparations of antihemophilic factor are held together by noncovalent bonds.

THE HETEROGENEITY OF GAMMA-GLOBULIN IN POST-EXERCISE URINE

1964 ◽  
Vol 42 (7) ◽  
pp. 1065-1097 ◽  
Author(s):  
M. H. Freedman ◽  
G. E. Connell
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Polypeptide Chain ◽  
Gel Filtration ◽  
Low Molecular Weight ◽  
Starch Gel Electrophoresis ◽  
Single Individual ◽  
Post Exercise ◽  
Γ Globulins ◽  
Normal Urine

Post-exercise urine was collected and the protein was precipitated with ammonium sulphate. The γ-globulin was separated from other urinary proteins by preparative starch block electrophoresis. The γ-globulin was then separated by gel filtration into two fractions, the faster one consisting mainly of 7 S γ-globulin while the slower one contained "low molecular weight" γ-globulin sedimenting at 3.5 S. The low molecular weight γ-globulins were further fractionated on CM-Sephadex to yield four fractions. Each of the chromatographic fractions was shown to be heterogeneous by starch gel electrophoresis at pH 4. The same degree of heterogeneity was observed in preparations of pooled urinary γ-globulin and γ-globulin from a single individual. The two major electrophoretic components of each chromatographic fraction formed precipitates in agar diffusion tests with antiserum to 7 S γ-globulin and to the L-polypeptide chain of 7 S γ-globulin. Two chromatographic fractions reacted with antiserum to the H-polypeptide chain of 7 S γ-globulin. All of the fractions failed to react with antiserum to β2A-globulin and β2M-globulin. Amino acid analysis showed distinct differences among the chromatographic fractions. One of the fractions closely resembled the L-chain of 7 S plasma γ-globulin with respect to amino acid composition. After reduction of disulphide bonds and alkylation most of the urinary γ-globulin resembles L-chain in electrophoretic behavior. The γ-globulins of post-exercise urine were found to be qualitatively similar to the γ-globulins of normal urine but post-exercise γ-globulins were present quantitatively in much larger amounts.

Permeability Enhancing and Chemotactic Activities of Lower Molecular Weight Degradation Products of Human Fibrinogen

1981 ◽  
Vol 45 (01) ◽  
pp. 090-094 ◽  
Author(s):  
Katsuo Sueishi ◽  
Shigeru Nanno ◽  
Kenzo Tanaka
Keyword(s):  
Molecular Weight ◽  
Degradation Products ◽  
Electron Microscopic Observation ◽  
Chemotactic Activity ◽  
Lower Molecular Weight ◽  
Electron Microscopic ◽  
Human Fibrinogen ◽  
Rabbit Skin ◽  
Molecular Weights ◽  
Active Fragments

SummaryFibrinogen degradation products were investigated for leukocyte chemotactic activity and for enhancement of vascular permeability. Both activities increased progressively with plasmin digestion of fibrinogen. Active fragments were partially purified from 24 hr-plasmin digests. Molecular weights of the permeability increasing and chemotactic activity fractions were 25,000-15,000 and 25,000 respectively. Both fractions had much higher activities than the fragment X, Y, D or E. Electron microscopic observation of the small blood vessels in rabbit skin correlated increased permeability with the formation of characteristic gaps between adjoining endothelial cells and their contraction.These findings suggest that lower molecular weight degradation products of fibrinogen may be influential in contributing to granulocytic infiltration and enhanced permeability in lesions characterized by deposits of fibrin and/or fibrinogen.

In Vivo Studies on the Inhibition of Coagulation by Fractionated Heparin and by a Heparin Analogue I. Effects of Heparin Fractions

1981 ◽  
Vol 46 (03) ◽  
pp. 612-616 ◽  
Author(s):  
U Schmitz-Huebner ◽  
L Balleisen ◽  
F Asbeck ◽  
J van de Loo
Keyword(s):  
Molecular Weight ◽  
Day Treatment ◽  
Gel Filtration ◽  
Weight Fraction ◽  
In Vivo Studies ◽  
Low Molecular Weight ◽  
High Molecular Weight Fraction ◽  
Platelet Factor ◽  
Heparin Fractions

SummaryHigh and low molecular weight heparin fractions obtained by gel filtration chromatography of sodium mucosal heparin were injected subcutaneously into six healthy volunteers and compared with the unfractionated substance in a cross-over trial. Equal doses of 5,000 U were administered twice daily over a period of three days and heparin activity was repeatedly controlled before and 2, 4, 8 hrs after injection by means of the APTT, the anti-Xa clotting test and a chromogenic substrate assay. In addition, the in vivo effect of subcutaneously administered fractionated heparin on platelet function was examined on three of the volunteers. The results show that s.c. injections of the low molecular weight fraction induced markedly higher anti-Xa activity than injections of the other preparations. At the same time, APTT results did not significantly differ. Unfractionated heparin and the high molecular weight fraction enhanced ADP-induced platelet aggregation and collagen-mediated MDA production, while the low molecular weight fraction hardly affected these assays, but potently inhibited thrombin-induced MDA production. All heparin preparations stimulated the release of platelet Factor 4 in plasma. During the three-day treatment periods, no side-effects and no significant changes in the response to heparin injections were detected.

scholarly journals The complete amino acid sequence of the low molecular weight cytosolic acid phosphatase

1989 ◽  
Vol 264 (5) ◽  
pp. 2560-2567
Author(s):  
G Camici ◽  
G Manao ◽  
G Cappugi ◽  
A Modesti ◽  
M Stefani ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Acid Phosphatase ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Low Molecular Weight ◽  
Complete Amino Acid Sequence

CLEAVAGE PATHWAY,AND SPECIFICITY OF LEUCOCYTE ELASTASE AS COMPARED TO PLASMIN DURING FIBRINOGEN DEGRADATION

1987 ◽  
Author(s):  
L Goretzki ◽  
E Miller ◽  
A Henschen
Keyword(s):  
Amino Acid ◽  
Time Course ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Reversed Phase ◽  
Cleavage Sites ◽  
Human Fibrinogen ◽  
Leucocyte Elastase ◽  
N Terminus

Plasmin and leucocyte elastase are regarded as the two medically most important fibrin(ogen)-degrading proteolytic enzymes. There is, however, a considerable difference in information available about the cleavage specificities and fragmentation pathways of these two enzymes. Degradation by plasmin has been studied already for a long time in great detail so that now the time course of the degradation, the cleavage sites and the functional properties of many fragments are well known. In contrast, relatively little is known about the degradation by leucocyte elastase, except that the overall cleavage pattern resembles that obtained with plasminIn this investigation the leucocyte elastase-mediated degradation of fibrinogen has been examined by means of proteinchemi-cal methods. Human fibrinogen was incubated with human enzyme material for various periods of time and at some different enzyme concentrations. The split products formed at the various stages were isolated in pure form by gel filtration followed by reversed-phase high-performance liquid chromatography. The fragments were identified by N-terminal amino acid sequence and amino acid composition. The course of the degradation was also monitored by sodium dodecylsulfate-polyacrylamide gel electrophoresis. All cleavage patterns were compared with the corresponding patterns from plasmic degradation. It could be confirmed that X-, D- and E-like fragments are formed also with elastase. However, several early elastolytic Aα-chain fragments are characteristically different from plasmic fragments. The previously identified N-terminal cleavage site in the Aα-chain, i.e. after position 21, was found to be the most important site in this region of fibrinogen. The very early degradation of the Aα-chain N-terminus by elastase is in strong contrast to the stability against plasmin. Several cleavage sites in N-terminal region of the Bβ-chain were observed, though the low amino acid specificity of elastase partly hampered the identification. The γ-chain N-terminus was found to be as highly stable towards elastase as towards plasmin. The results are expected to contribute to the understanding of the role of leucocyte elastase in pathophysiologic fibrino(geno)lysis

Characterization of an isozyme of S-adenosylmethionine synthetase from rat liver

1984 ◽  
Vol 62 (5) ◽  
pp. 276-279 ◽  
Author(s):  
C. H. Lin ◽  
W. Chung ◽  
K. P. Strickland ◽  
A. J. Hudson
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Enzymatic Synthesis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Aromatic Amino Acids ◽  
Adenosylmethionine Synthetase ◽  
Cellulose Column

An isozyme of S-adenosylmethionine synthetase has been purified to homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and gel filtration on a Sephadex G-200 column. The purified enzyme is very unstable and has a molecular weight of 120 000 consisting of two identical subunits. Amino acid analysis on the purified enzyme showed glycine, glutamate, and aspartate to be the most abundant and the aromatic amino acids to be the least abundant. It possesses tripolyphosphatase activity which can be stimulated five to six times by S-adenosylmethionine (20–40 μM). The findings support the conclusion that an enzyme-bound tripolyphosphate is an obligatory intermediate in the enzymatic synthesis of S-adenosylmethionine from ATP and methionine.

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