Increased Yield of Human Tissue-Type Plasminogen Activator Obtained by Means of Recombinant DNA Technology

Browne, Michael; Dodd, Ian B.; Carey, J.E.; Chapman, C.G.; Robinson, Jan

doi:10.1055/s-0038-1657864

Cited by 28 publications

(9 citation statements)

References 12 publications

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“…geneity from the culture supernatant of a genetically manipulated human cell line (Browne et al, 1985) and had M, 63 000 by SDS/PAGE/fibrin-overlay autography (Granelli-Piperno & Reich, 1978). Human thrombin (1000 NIH units/mg of protein) was obtained from Sigma Chemical Co. (Poole, Dorset, U.K.).…”

Section: Materials and Methods Materialsmentioning

confidence: 99%

The interaction of streptokinase.plasminogen activator complex, tissue-type plasminogen activator, urokinase and their acylated derivatives with fibrin and cyanogen bromide digest of fibrinogen. Relationship to fibrinolytic potency in vitro

Cassels¹,

Fears²,

Smith³

1987

Biochemical Journal

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The effects of purified soluble fibrin and of fibrinogen fragments (fibrin mimic) on the activation of Lys-plasminogen (i.e. plasminogen residues 77-790) to plasmin by streptokinase.plasminogen activator complex and by tissue-type plasminogen activator were studied. Dissociation constants of both activators were estimated to lie in the range 90-160 nM (fibrin) and 16-60 nM (CNBr-cleavage fragments of fibrinogen). The kinetic mechanism for both types of activator comprised non-essential enzyme activation via a Rapid Equilibrium Ordered Bireactant sequence. In order to relate the fibrin affinity of plasminogen activators to their fibrinolytic potency, the rate of lysis of supported human plasma clots formed in the presence of unmodified or active-centre-acylated precursors of plasminogen activators was studied as a function of the concentration of enzyme derivative. The concentrations of unmodified enzyme giving 50% lysis/h in this assay were 0.9, 2.0 and 11.0 nM for tissue-type plasminogen activator, streptokinase.plasmin(ogen) and urokinase respectively. However, the potencies of active-centre-acylated derivatives of these enzymes suggested that acylated-tissue plasminogen activator and streptokinase.plasminogen complexes of comparable hydrolytic stability were of comparable potency. Both types of acyl-enzyme were significantly more potent than acyl-urokinases.

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Section: Materials and Methods Materialsmentioning

confidence: 99%

The interaction of streptokinase.plasminogen activator complex, tissue-type plasminogen activator, urokinase and their acylated derivatives with fibrin and cyanogen bromide digest of fibrinogen. Relationship to fibrinolytic potency in vitro

Cassels¹,

Fears²,

Smith³

1987

Biochemical Journal

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“…The recombinant Bowes melanoma cell line TRBM 6 (6) was kindly supplied by Dr M. J. Browne Pharmacia (Great Britain) Ltd., Milton Keynes, U. K. Bovine serum albumin and poly-D-lysine (M, : 100,000) were from Sigma, Poole, U. K.…”

Section: Methodsmentioning

confidence: 99%

Isolation and Preliminary Characterisation of Active B-Chain of Recombinant Tissue-Type Plasminogen Activator

1986

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SummaryPurified 2-chain recombinant tissue-type plasminogen activator (t-PA) was reduced under mild conditions - 10 mM dithiothreitol/ 5° C/1.5 h - and the two chains were separated by chromatography on lysine Sepharose. The t-PA B chain was fully active as determined by its activity towards the chromogenic substrate S-2288 (H-D-ile-pro-arg p-nitroanilide). Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing or non-reducing conditions revealed a single polypeptide at Mr = 35,000 or 29,000 respectively. In addition, under non-reducing conditions a fibrinolytic band at apparent Mr = 29,000 was present after fibrin zymography. The N-terminal sequence was confirmed as ile-lys-gly. The t-PA B chain had a specific amidolytic activity, using S-2288, of 170,000 to 210,000 SU/mg protein. (This compares to a specific activity of the native 2-chain t-PA of 170,000 SU/mg). It resembles urokinase-type plasminogen activator in its inability to be stimulated by fibrin and its dose response on human fibrin plates. However, t-PA B-chain was stimulated to almost the same extent as t-PA by poly-D-lysine. The isoelectric points, at pH 5.6 and 5.7, fall outside the range generally quoted for t-PA preparations (pH 7.8-8.8).

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“…The synthesis and characterization of the plasmin Achain/t-PA B-chain hybrid (apparent Mr 86000) and the plasmin A-chain/u-PA B-chain hybrid (apparent Mr 86000) were described elsewhere (Robinson et al, 1988). t-PA was purified from the recombinant Bowes-melanoma cell line TRBM6 (Browne et al, 1985;Dodd et al, 1986b); the major species had an apparent Mr of 63000. The origin and purification of the other proteins used, and the preparation of soluble fibrin by digestion of clotted human fibrin using NaBr, have been described previously (Fears et al, 1985).…”

Section: Materials and Methods Materialsmentioning

confidence: 99%

Kinetic studies on novel plasminogen activators. Demonstration of fibrin enhancement for hybrid enzymes comprising the A-chain of plasmin (Lys-78) and B-chain of tissue-type plasminogen activator (Ile-276) or urokinase (Ile-159)

Fears

Dodd

Ferres

et al. 1990

Biochemical Journal

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The activation of plasminogen by two novel hybrid enzymes, constructed from the A-chain of plasmin and the B-chains of tissue-type plasminogen activator (t-PA) or urokinase, was compared with the activation by the parent enzymes. Basal kinetic constants for 'Lys-plasminogen' (human plasminogen with N-terminal lysine) and 'Glu-plasminogen' (human plasminogen with N-terminal glutamic acid) activation were similar to those of the parent activators. The Km for plasminogen turnover for both hybrid enzymes was considerably decreased in the presence of both soluble fibrin and a mimic, a CNBr digest of fibrinogen. These enhancements and the related apparent negative co-operativity are similar to the behaviour of t-PA itself. The results are discussed with regard to the molecular features involved in the mechanism of fibrin stimulation.

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Increased Yield of Human Tissue-Type Plasminogen Activator Obtained by Means of Recombinant DNA Technology

Cited by 28 publications

References 12 publications

The interaction of streptokinase.plasminogen activator complex, tissue-type plasminogen activator, urokinase and their acylated derivatives with fibrin and cyanogen bromide digest of fibrinogen. Relationship to fibrinolytic potency in vitro

The interaction of streptokinase.plasminogen activator complex, tissue-type plasminogen activator, urokinase and their acylated derivatives with fibrin and cyanogen bromide digest of fibrinogen. Relationship to fibrinolytic potency in vitro

Isolation and Preliminary Characterisation of Active B-Chain of Recombinant Tissue-Type Plasminogen Activator

Kinetic studies on novel plasminogen activators. Demonstration of fibrin enhancement for hybrid enzymes comprising the A-chain of plasmin (Lys-78) and B-chain of tissue-type plasminogen activator (Ile-276) or urokinase (Ile-159)

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