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Effects of N-glycosylation on in vitro activity of Bowes melanoma and human colon fibroblast-derived tissue plasminogen activator

Tissue-type plasminogen activator (t-PA), when isolated from human colon fibroblast (hcf) cells, is N-glycosylated differently than when isolated from the Bowes melanoma (m) cell line (Parekh et al., 1988). Both hcf- and m-t-PA can be separated into type I t-PA (with three occupied N-glycosylation s...

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Published in:Biochemistry (Easton) 1989-09, Vol.28 (19), p.7662-7669
Main Authors: Wittwer, Arthur J, Howard, Susan C, Carr, Linda S, Harakas, Nikos K, Feder, Joseph, Parekh, Raj B, Rudd, Pauline M, Dwek, Raymond A, Rademacher, Thomas W
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container_end_page 7669
container_issue 19
container_start_page 7662
container_title Biochemistry (Easton)
container_volume 28
creator Wittwer, Arthur J
Howard, Susan C
Carr, Linda S
Harakas, Nikos K
Feder, Joseph
Parekh, Raj B
Rudd, Pauline M
Dwek, Raymond A
Rademacher, Thomas W
description Tissue-type plasminogen activator (t-PA), when isolated from human colon fibroblast (hcf) cells, is N-glycosylated differently than when isolated from the Bowes melanoma (m) cell line (Parekh et al., 1988). Both hcf- and m-t-PA can be separated into type I t-PA (with three occupied N-glycosylation sequons, at Asn-117, -184, and -448) and type II t-PA (with two occupied sequons, at Asn-117 and -448). Oligosaccharide analysis of each of these types of t-PA indicates that hcf-t-PA and m-t-PA have no glycoforms in common, despite having the same primary amino acid sequence. We have therefore compared in vitro the enzymatic activities and fibrin binding of type I and type II hcf- and m-t-PA with those of aglycosyl t-PA isolated from tunicamycin-treated cells. Plasminogen activation kinetics were determined by using an indirect amidolytic assay with Glu-plasminogen and a chromogenic plasmin substrate. In the absence of stimulator, there was little difference in activity between type I and type II t-PA, but the activity of aglycosyl t-PA was 2-4-fold higher than that of the corresponding glycosylated t-PA. In the presence of a fibrinogen fragment stimulator, the Kcat value of type II t-PA was approximately 5-fold that of type I t-PA from the same cell line, while the Km values for activation of Glu-plasminogen were similar (0.13-0.18 microM). The stimulated activity of glycosyl t-PA was similar to that of type II t-PA.
doi_str_mv 10.1021/bi00445a022
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Psychology</topic><topic>General aspects, investigation methods, hemostasis, fibrinolysis</topic><topic>Glycosylation</topic><topic>Humans</topic><topic>man</topic><topic>Melanoma - pathology</topic><topic>Molecular and cellular biology</topic><topic>Plasminogen - metabolism</topic><topic>Tissue Plasminogen Activator - metabolism</topic><topic>Tumor Cells, Cultured</topic><topic>Tunicamycin - pharmacology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Wittwer, Arthur J</creatorcontrib><creatorcontrib>Howard, Susan C</creatorcontrib><creatorcontrib>Carr, Linda S</creatorcontrib><creatorcontrib>Harakas, Nikos K</creatorcontrib><creatorcontrib>Feder, Joseph</creatorcontrib><creatorcontrib>Parekh, Raj B</creatorcontrib><creatorcontrib>Rudd, Pauline M</creatorcontrib><creatorcontrib>Dwek, Raymond A</creatorcontrib><creatorcontrib>Rademacher, Thomas W</creatorcontrib><collection>Istex</collection><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biochemistry Abstracts 3</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wittwer, Arthur J</au><au>Howard, Susan C</au><au>Carr, Linda S</au><au>Harakas, Nikos K</au><au>Feder, Joseph</au><au>Parekh, Raj B</au><au>Rudd, Pauline M</au><au>Dwek, Raymond A</au><au>Rademacher, Thomas W</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Effects of N-glycosylation on in vitro activity of Bowes melanoma and human colon fibroblast-derived tissue plasminogen activator</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>1989-09-19</date><risdate>1989</risdate><volume>28</volume><issue>19</issue><spage>7662</spage><epage>7669</epage><pages>7662-7669</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>Tissue-type plasminogen activator (t-PA), when isolated from human colon fibroblast (hcf) cells, is N-glycosylated differently than when isolated from the Bowes melanoma (m) cell line (Parekh et al., 1988). Both hcf- and m-t-PA can be separated into type I t-PA (with three occupied N-glycosylation sequons, at Asn-117, -184, and -448) and type II t-PA (with two occupied sequons, at Asn-117 and -448). Oligosaccharide analysis of each of these types of t-PA indicates that hcf-t-PA and m-t-PA have no glycoforms in common, despite having the same primary amino acid sequence. We have therefore compared in vitro the enzymatic activities and fibrin binding of type I and type II hcf- and m-t-PA with those of aglycosyl t-PA isolated from tunicamycin-treated cells. Plasminogen activation kinetics were determined by using an indirect amidolytic assay with Glu-plasminogen and a chromogenic plasmin substrate. In the absence of stimulator, there was little difference in activity between type I and type II t-PA, but the activity of aglycosyl t-PA was 2-4-fold higher than that of the corresponding glycosylated t-PA. In the presence of a fibrinogen fragment stimulator, the Kcat value of type II t-PA was approximately 5-fold that of type I t-PA from the same cell line, while the Km values for activation of Glu-plasminogen were similar (0.13-0.18 microM). The stimulated activity of glycosyl t-PA was similar to that of type II t-PA.</abstract><cop>Washington, DC</cop><pub>American Chemical Society</pub><pmid>2514792</pmid><doi>10.1021/bi00445a022</doi><tpages>8</tpages></addata></record>
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ispartof Biochemistry (Easton), 1989-09, Vol.28 (19), p.7662-7669
issn 0006-2960
1520-4995
language eng
recordid cdi_proquest_miscellaneous_79424344
source ACS CRKN Legacy Archives
subjects Biological and medical sciences
Blood coagulation. Blood cells
cell lines
Cells, Cultured
Colon - cytology
Enzyme Activation - drug effects
Fibrin
Fibroblasts
Fundamental and applied biological sciences. Psychology
General aspects, investigation methods, hemostasis, fibrinolysis
Glycosylation
Humans
man
Melanoma - pathology
Molecular and cellular biology
Plasminogen - metabolism
Tissue Plasminogen Activator - metabolism
Tumor Cells, Cultured
Tunicamycin - pharmacology
title Effects of N-glycosylation on in vitro activity of Bowes melanoma and human colon fibroblast-derived tissue plasminogen activator
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