The effect of polymerised fibrin on the catalytic activities of one-chain tissue-type plasminogen activator as revealed by an analogue resistant to plasmin cleavage.
TL;DR: The results were interpreted to mean that fibrin binding can induce an activated state of the intact tPA one-chain form, also when plasmin is generated as a result of a plAsminogen activation reaction.
About: This article is published in Biochimica et Biophysica Acta.The article was published on 1988-01-01. It has received 72 citations till now. The article focuses on the topics: Plasmin & Plasminogen activator.
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TL;DR: This work has shown that the protease Nexin I antagonist acts as a “spatially aggregating force” to drive down the activity of the plasminogen during the courtship process.
Abstract: PLASMINOGEN ACTIVATOR INHIBITORS 104 Plasminogen Activator Inhibitor 1 104 Plasminogen Activator Inhibitor 2 106 Protease Nexin I .... .... 108 PHYSIOLOGICAL FUNCTIONS OF PLASMINOGEN ACTIVATION ........ 109 Matrix Breakdown 109 Cell Migration 111 Ovulation 113 CONCLUDING REMARKS 115
877 citations
TL;DR: One-chain u-PA has a variety of properties similar to the one-chain proenzyme forms of other serine proteases and that it should, therefore, be considered as a genuine proen enzyme form of U-PA.
241 citations
TL;DR: Variants of tPA were found that had reduced activity with respect to each tested property; in a few cases increased activity was observed, and a model of the tPA protease domain has been used to map some of the critical residues and regions.
217 citations
TL;DR: Bat-PA(H), the full-length form, is the only Bat-PA species which binds tightly to fibrin, although each of the three species exhibit remarkable stimulation by a fibrIn cofactor.
169 citations
162 citations
References
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TL;DR: This chapter describes methods for sodium dodecyl sulfate gel electrophoresis and the characterization of proteins separated on SDS gels, designed to alkylate various functional groups of the proteins so as to maximize the likelihood of irreversible denaturation.
Abstract: Publisher Summary This chapter describes methods for sodium dodecyl sulfate (SDS) gel electrophoresis and the characterization of proteins separated on SDS gels. SDS dissociates proteins into their constituent polypeptide chains. Polyacrylamide gel electrophoresis in the presence of SDS separates polypeptide chains according to their molecular weights. Thus, the size of the polypeptide chains of a given protein can be determined by comparing their electrophoretic mobilities on SDS gels to the mobilities of marker proteins with well-characterized polypeptide chain molecular weights. SDS-polyacrylamide gel electrophoresis is easily and rapidly performed, requires only inexpensive equipment, and can be used with microgram amounts of protein. The technique is both reliable and reproducible, and the results are easy to interpret. The procedure described in the chapter is designed to alkylate various functional groups of the proteins so as to maximize the likelihood of irreversible denaturation. Small changes in electrophoretic mobility may occur after such extensive alkylation.
1,632 citations
TL;DR: The kinetic analysis suggested that the activation in the presence of fibrin occurs through binding of an activator molecule to the clot surface and subsequent addition of plasminogen (sequential ordered mechanism) to form a cyclic ternary complex.
1,223 citations
TL;DR: Bacterial clones containing human tissue-type plasminogen activator cDNA sequences were identified in a cDNA library prepared using gel-fractionated mRNA from human melanoma cells and a polypeptide was produced having the fibrinolytic properties characteristic of authentic human t-PA.
Abstract: Bacterial clones containing human tissue-type plasminogen activator (t-PA) cDNA sequences were identified in a cDNA library prepared using gel-fractionated mRNA from human melanoma cells. A plasmid containing the Escherichia coli trp promoter and the cDNA sequence coding f or the 527-amino acid mature t-PA protein was constructed for expression in E. coli. A polypeptide was produced having the fibrinolytic properties characteristic of authentic human t-PA.
1,219 citations
01 Jan 1981
TL;DR: In this paper, the activation of Gluplasminogen and Lys-minminogen in the presence of fibrinogen (f) was studied in purified systems, and the initial rate of activation (u) was calculated.
Abstract: The kinetics of the activation of Glu-plasminogen and Lys-plasminogen (P) by a two-chain form of human tissue plasminogen activator (A) were studied in purified systems, and in the presence of fibrinogen (f) and of fibrin films (F) of increasing size and surface density. The activation in the purified systems followed Michaelis-Menten kinetics with a Michaelis constant of 65 p~ and a catalytic rate constant of 0.06 s-' for Glu-plasminogen as compared to 19 p~ and 0.2 s" for Lysplasminogen. In the presence of fibrinogen plots of l/u uersus 1/[P] or l/u uersus l/[fl yielded straight lines with an apparent Michaelis constant at infinite [fl of 28 PM and a catalytic rate constant of 0.3 s-' for Gluplasminogen as compared to 1.8 p~ and 0.3 s-' for Lysplasminogen. In the systems with fibrin, plasmin was estimated from the rate of release of '"I from "'I-labeled fibrin films. The initial rate of activation (u) was calculated and Lineweaver-Burk plots of l/u uersus 1/ [PI or l/u uersus l/[F] yielded straight lines. Activation occurred with an intrinsic Michaelis constant of 0.16 PM and a catalytic rate constant of 0.1 s-' for Gluplasminogen as compared to 0.02 PM and 0.2 s" for Lysplasminogen. The kinetic analysis suggested that the activation in the presence of fibrin occurs through binding of an activator molecule to the clot surface and subsequent addition of plasminogen (sequential ordered mechanism) to form a cyclic ternary complex. The low Michaelis constant in the presence of fibrin allows efficient plasminogen activation on a fibrin clot, while its high value in the absence of fibrin prevents efficient activation in plasma.
1,149 citations
TL;DR: This paper presents a versatile and efficient procedure for the construction of oligodeoxyribonucleotide directed site-specific mutations in DNA fragments cloned into M13 derived vectors, and production of a transition mutation in a clone of the yeast MATa1 gene is described.
Abstract: This paper presents a versatile and efficient procedure for the construction of oligodeoxyribonucleotide directed site-specific mutations in DNA fragments cloned into M13 derived vectors. As an example, production of a transition mutation in a clone of the yeast MATa1 gene is described. The oligonucleotide is hybridized to the template DNA and covalently closed closed double stranded molecules are generated by extension of the oligonucleotide primer with E. coli DNA polymerase (large fragment) and ligation with T4 DNA ligase. The resulting double stranded closed circular DNA (CC-DNA) is separated from unligated and incompletely extended molecules by alkaline sucrose gradient centrifugation. This purification is essential for production of mutants at high efficiency. Competent E. coli JM101 cells are transformed with the CC-DNA fraction and single stranded DNA is isolated from individual plaques. The recombinants are screened for mutant molecules by 1) restriction endonuclease screening for the loss of the Hinf I site in the target region, and 2) by dot blot hybridization using the mutagenic oligonucleotide as probe. Double stranded DNA is isolated from the sequencing. Efficiency of mutant production is in the range of 10-45% and no precautions to prevent mismatch repair are required.
1,011 citations