A Refined Kinetic Analysis of Plasminogen Activation by Recombinant Bovine Tissue-Type Plasminogen Activator Indicates Two Interconvertible Activator Forms†
Summary (2 min read)
- The activation of plasminogen is an important process associated with degradation of an extracellular matrix such as dissolving of blood clots, tissue remodeling, and invasive growth of cancer cells.
- Tissue-type plasminogen activator (tPA)1 and urokinase-type plasminogen activator (uPA) are the two major proteins responsible for conversion of plasminogen to plasmin.
- The tPA-induced process is stimulated significantly on the surface of fibrin, and tPA is regarded as the fibrinolytic activator.
- Kinetic analysis of the human tPA catalysis was rendered in a number of papers (7-9) and resulted in several conclusions concerning its mechanism.
- This work is part of the FØTEK program supported by the Danish Dairy Research Foundation (Danish Dairy Board) and the Danish Government.
MATERIALS AND METHODS
- P. pastorisGS115 (his4) (10), the protease deficient strain SMD1168 (his4, pep4), and the expression vectors pPIC9K and pHIL-D2 were purchased from Invitrogen Corp.
- Sequencing, ligation, transformation ofEscherichia coli, DNA preparation, PCR, and other DNA modifying processes were performed according to the manufacturers’ recommendations or standard laboratory procedures.
- To generate the native N-terminus of tPA after cleavage by the signal peptidases , site specific mutagenesis was performed on pPIC9K/tPA, using the primer 5′-CTCGAGAAAAGAGAGGCTGAAGCTTCGTACAAAGTGACCTGCAGAGAT-3′.
- The plasminogen activation potential of tPA was evaluated in a coupled peptidyl anilide assay, where the formation of plasmin was measured by its hydrolysis of the chromogenic substrate S-2251.
- A more complex scheme, described in model 2, considers the existence of a prestationary step before the above reactions which distorts linearity of the plot in its initial part.
- The expression level of bovine tPA was optimized by a 3-fold strategy: (1) improving the secretion, (2) increasing the transcription of the tPA gene, and (3) minimizing the proteolytic breakdown of tPA in the expression media.
- Second,∼10 000 colonies were screened on YPD plates containing different concentrations of G418 for multicopy colony selection.
- The band at approximately 50 kDa turned out to be the proteinase domain, even though it was 10-20 kDa higher than the calculated mass (29 kDa).
- (2) The reaction was performed with a decreased concentration of tPA (1 × 10-3 µM) in the presence of fibrinogen fragments.
- At the same time, analysis of the prestationary kinetics from the same experiment revealed the presence of a tPA form with high affinity to Pg being in equilibrium with a low affinity form, see model 2.
- The authors have produced recombinant bovine tissue-type plasminogen activator in the yeast,P. pastoris.
- The equilibrium between these forms in the preparation was shifted in favor of tPA* (g50:1).
- At the same time, it showed a≈25-fold increase ofkcat, when compared to the Fb-free tPA.
- The mechanism of activation of human tPA by fibrinogen fragments is presumably the same as for bovine tPA according to the values of calculated rate constants.
- To characterize other properties of the produced tPA, inhibition of bovine single-chain tPA by PAI-1 and PAI-2 was investigated.
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Q1. What contributions have the authors mentioned in the paper "A refined kinetic analysis of plasminogen activation by recombinant bovine tissue-type plasminogen activator indicates two interconvertible activator forms†" ?
The bovine single-chain tPAmediated activation of bovine plasminogen was studied in the presence and absence of fibrinogen fragments. The authors have proposed a refined new method of kinetic analysis which allows examination of both stationary and prestationary phases of this process. The activation of plasminogen has been studied in detail in the human system from where the involved protein components have been identified and characterized. Bovine mastitis is an inflammatory disease of the mammary gland induced by various microorganisms, and a 20-fold increase in tPA activity has been reported in the milk of cows infected with Staphylococcus aureus ( 1 ). The activation of plasminogen by tPA is greatly increased by the Rs2-casein dimer ( 2 ), and in order to study this system in more detail it is necessary to obtain bovine tPA.