K. Laki

The polymerization of proteins: the action of thrombin on fibrinogen

Abstract Viscosity measurements and ultracentrifugal experiments show no difference between fibrinogen and fibrinogen incubated with thrombin, both kept at pH 4.85, although the progressing action of thrombin can be demonstrated.

The amino acid composition of actin, myosin, tropomyosin and the meromyosins

Abstract The amino-acid composition of actin, tropomyosin, myosin, L-meromyosin, and H-meromyosin has been determined by the Moore and Stein Chromatographic technique. The data obtained on the meromyosins when added up yield the amino acid composition of myosin. Tropomyosin and actin taken in equal proportion also approximate myosin, although the agreement with some of the amino acids is poor. Although not permitting the conclusion that myosin is a compound of actin and tropomyosin, the data strongly suggest a relationship, and do not rule out the possibility that actin and tropomyosin may be subunits in the myosin molecule.

The amino acid composition of bovine prothrombin

Abstract The full amino acid composition of bovine prothrombin has been measured by using ion-exchange chromatography. In addition, other methods were used for the estimation of tyrosine, tryptophan, phenylalanine, and amide ammonia. Cysteine-cystine were estimated as cysteic acid. Eighteen amino acids and hexosamine are represented, glutamic acid, aspartic acid, and arginine being present in highest weight percentage. There is no resemblance between the known amino acid composition of serum albumin and that of prothrombin, though the two proteins are closely related with regard to their physicochemical characteristics in many respects.

Light scattering studies on the clotting of fibrinogen.

Summary The light-scattering method was applied to study the clotting of purified bovine fibrinogen. The molecular weight of fibrinogen was found to be 540,000 and the length 840 A. Fibrin dissolved in urea and guanidine gave the same molecular weight as fibrinogen under similar conditions. It was found that during clotting of fibrinogen larger and larger particles were built up through side-by-side and end-to-end associations of the apparently original fibrinogen particles. The relative importance of the two kinds of associations depends on pH and ionic strength. The arrest of the polymerization and a subsequent depolymerization of the polymerized particles revealed that the end-to-end associations are weaker than the side-by-side assocations. The clotting of fibrinogen by papain was found to be similar to the clotting brought about by thrombin.

Studies on the physiological activity of the peptide released during the fibrinogen-fibrin conversion

Abstract The thrombin (EC 3.4.4.13) catalyzed conversion of fibrinogen to fibrin results in the release of several peptides. The peptides have been isolated from the interaction of human thrombin with human fibrinogen. Two of the acidic peptides (α and β) released from human fibrinogen contain 16 amino acids each and differ only in that the α-peptide containis a phosphate group covalently bound to a serine residue. The β-peptide is physiologically active in that it possesses the ability to potentiate the bradykinin-induced contraction of rat-uterus muscle. The α-peptide is inactive when tested in a similar manner. Structural modifications may be performed on the β-peptide without loss of physiological activity. This acitivity is not diminished by the removal of the carboxyl end-group or by a selective enzymic cleavage of the peptide. It is suggested that the β-peptide, which is released during the blood-clotting process, may play a vital role in the regulation of the microcirculation of the blood.

The fragmentation of actin by thrombin. Isolation and characterization of the split products

Abstract Thrombin, a limited protease, hydrolyzes three bonds in actin at Arg-28, Arg-39, and Lys-113, thereby producing two smaller peptides and two larger fragments. The location of the bonds split was identified in the amino acid sequence of actin by isolating the split products and carrying out amino acid analysis, and N- and C-terminal determinations.

THE MODE OF ACTION OF THE LAKI-LORAND FACTOR IN THE CLOTTING OF FIBRINOGEN.

Abstract The thrombin-induced clotting of fibrinogen does not lead to hemostasis ( Duckert et al, 1960 ). The fibrin molecules in the clot are connected only with hydrogen bonds, and are readily dispersed in concentrated urea solutions ( Laki and Lorand 1948 ; Laki and Chandrasekhar, 1963 ). It was discovered by Laki and Lorand (1948) that the intervention of a plasma component (called: Laki-Lorand Factor = LLF, Fibrin Stabilizing Factor = FSF, Fibrinase) in the presence of Ca-ions was needed to render the clot insoluble in urea solutions. The plasma component introduced bonds between the fibrin molecules so that urea could not disperse the clot. A few years ago, Lowey et al (1961) succeeded in purifying this plasma component and showed that it was an enzyme. The purpose of this communication is to show that it is now possible to describe the mechanism by which the primary bonds are introduced between the fibrin molecules.

The minimal molecular weight of actin estimated with the use of carboxypeptidase A

Abstract The minimal molecular weight of actin has been determined by the release of the COOH-terminal group, phenylalanine. A value of 50,000 ± 10% was obtained.

Nucleic acid and nucleotide content of myosin preparations

Abstract Myosin preparations contain 0.5–0.8% of ribonucleic acid, which cannot be separated from the protein by the usual purification procedures, but can be extracted with hot 10 % NaCl solution and also removed partly after digestion with ribonuclease. The physicochemical and enzymic properties of myosin are unaffected by the depolymerization of the nucleic acid by ribonuclease. Hot TCA extracts from myosin contained the hydrolysis products of the nucleic acid, and an extra amount of adenine. The amount of the extra adenine is small in myosin A, but amounts to 0.1–0.4 group/105 g. protein in myosin B preparations. After extraction of the nucleic acid, the residual phosphorus amounts to 0.5–0.8 mole/105 g. protein.

On the interaction of F-actin with fibrin

Abstract It was found that F-actin combined with fibrin but not with fibrinogen. At saturating concentrations, two actin molecules were found to combine with one fibrin molecule. These experiments also revealed that under certain conditions thrombin was capable of fragmenting actin.