Abstract
We have (a) screened a variety of cell lines and body fluids for plasminogen activators and (b) studied the activity of proteases bound to α2- macroglobulin after exposing the complexes to partial degradation and/or denaturing procedures to unmask proteolytic activity. The respective results show (a) that the plasminogen activators in urine and cell culture media are generally of lower molecular weight than those in plasma; and (b) that proteases bound to α2-macroglobulin recover the ability to attack macromolecular substrates after exposure to sodium dodecyl sulfate while retaining the electrophoretic mobility of the protease inhibitor complex. This indicates that the protease and inhibitor are probably linked by covalent bonds. In contrast, other complexes formed between proteases and inhibitors of lower molecular weight (such as soybean or Kunitz inhibitors) are fully dissociated by sodium dodecyl sulfate (SDS). The experiments described were based on a new procedure for detecting proteolytic enzyme activity in SDS-polyacrylamide gels. The method relies on solutions of nonionic detergents for extracting SDS, after which the electrophoretic gel is applied to an indicator gel consisting of a fibrin- agar mixture. The method is sensitive, permitting the detection of proteinases in less than 1 μl of fresh plasma, and it is effective for resolving small differences in molecular weight. The procedure can be quantitated and, with minor modifications appropriate to each particular system, it has been applied to a broad spectrum of serine enzymes and proenzymes, including some that function in the pathways of fibrinolysis, coagulation and kinin-generation. Other potential applications appear likely.
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- Alper C. A. Inherited structural polymorphism in human C2: evidence for genetic linkage between C2 and Bf. J Exp Med. 1976 Oct 1;144(4):1111–1115. doi: 10.1084/jem.144.4.1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Barrett A. J., Starkey P. M. The interaction of alpha 2-macroglobulin with proteinases. Characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism. Biochem J. 1973 Aug;133(4):709–724. doi: 10.1042/bj1330709. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Christman J. K., Acs G. Purification and characterization of a cellular fibrinolytic factor associated with oncogenic transformation: the plasminogen activator from SV-40-transformed hamster cells. Biochim Biophys Acta. 1974 Mar 27;340(3):339–347. doi: 10.1016/0005-2787(74)90279-2. [DOI] [PubMed] [Google Scholar]
- Converse C. A., Papermaster D. S. Membrane protein analysis by two-dimensional immunoelectrophoresis. Science. 1975 Aug 8;189(4201):469–472. doi: 10.1126/science.1154021. [DOI] [PubMed] [Google Scholar]
- Deutsch D. G., Mertz E. T. Plasminogen: purification from human plasma by affinity chromatography. Science. 1970 Dec 4;170(3962):1095–1096. doi: 10.1126/science.170.3962.1095. [DOI] [PubMed] [Google Scholar]
- Granelli-Piperno A., Vassalli J. D., Reich E. Secretion of plasminogen activator by human polymorphonuclear leukocytes. Modulation by glucocorticoids and other effectors. J Exp Med. 1977 Dec 1;146(6):1693–1706. doi: 10.1084/jem.146.6.1693. [DOI] [PMC free article] [PubMed] [Google Scholar]
- HELMKAMP R. W., GOODLAND R. L., BALE W. F., SPAR I. L., MUTSCHLER L. E. High specific activity iodination of gamma-globulin with iodine-131 monochloride. Cancer Res. 1960 Nov;20:1495–1500. [PubMed] [Google Scholar]
- Harpel P. C. Studies on human plasma alpha 2-macroglobulin-enzyme interactions. Evidence for proteolytic modification of the subunit chain structure. J Exp Med. 1973 Sep 1;138(3):508–521. doi: 10.1084/jem.138.3.508. [DOI] [PMC free article] [PubMed] [Google Scholar]
- LAKI K. The polymerization of proteins; the action of thrombin on fibrinogen. Arch Biochem Biophys. 1951 Jul;32(2):317–324. doi: 10.1016/0003-9861(51)90277-9. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- MOSESSON M. W. The preparation of human fibrinogen free of plasminogen. Biochim Biophys Acta. 1962 Feb 26;57:204–213. doi: 10.1016/0006-3002(62)91112-5. [DOI] [PubMed] [Google Scholar]
- Sinclair J. H., Brown D. D. Retention of common nucleotide sequences in the ribosomal deoxyribonucleic acid of eukaryotes and some of their physical characteristics. Biochemistry. 1971 Jul 6;10(14):2761–2769. doi: 10.1021/bi00790a017. [DOI] [PubMed] [Google Scholar]
- Unkeless J. C., Tobia A., Ossowski L., Quigley J. P., Rifkin D. B., Reich E. An enzymatic function associated with transformation of fibroblasts by oncogenic viruses. I. Chick embryo fibroblast cultures transformed by avian RNA tumor viruses. J Exp Med. 1973 Jan 1;137(1):85–111. doi: 10.1084/jem.137.1.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Unkeless J., Dano K., Kellerman G. M., Reich E. Fibrinolysis associated with oncogenic transformation. Partial purification and characterization of the cell factor, a plasminogen activator. J Biol Chem. 1974 Jul 10;249(13):4295–4305. [PubMed] [Google Scholar]
- Vassalli J. D., Granelli-Piperno A., Griscelli C., Reich E. Specific protease deficiency in polymorphonuclear leukocytes of Chédiak-Higashi syndrome and beige mice. J Exp Med. 1978 Apr 1;147(4):1285–1290. doi: 10.1084/jem.147.4.1285. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vassalli J. D., Hamilton J., Reich E. Macrophage plasminogen activator: modulation of enzyme production by anti-inflammatory steroids, mitotic inhibitors, and cyclic nucleotides. Cell. 1976 Jun;8(2):271–281. doi: 10.1016/0092-8674(76)90011-8. [DOI] [PubMed] [Google Scholar]