Neutralization of bleomycin hydrolase by an epitope-specific antibody
Bleomycin hydrolase (BH) is a cysteine proteinase that terminates the pharmacological action of bleomycin (BLM). Amino acid sequence data obtained from a tryptic digest fragment of purified rabbit lung BH were used to synthesize a 14-amino acid peptide (LAVLEQEPIVLPAK; BHP14), which was conjugated t...
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| Veröffentlicht in: | Molecular pharmacology 1992-07, Vol.42 (1), p.57-62 |
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| creator | MORRIS, G MISTRY, J. S JANI, J. P MIGNANO, J. E SEBTI, S. M LAZO, J. S |
| description | Bleomycin hydrolase (BH) is a cysteine proteinase that terminates the pharmacological action of bleomycin (BLM). Amino acid
sequence data obtained from a tryptic digest fragment of purified rabbit lung BH were used to synthesize a 14-amino acid peptide
(LAVLEQEPIVLPAK; BHP14), which was conjugated to horseshoe crab hemocyanin and used to produce rabbit antiserum that was immunoreactive
to both BHP14 and rabbit BH. Anti-BHP14 binding to BHP14 could be competitively blocked by the presence of either BHP14 or
BH. Anti-BHP14 recognized both purified rabbit liver BH and postmicrosomal fraction from rabbit liver on Western blot, as
a single band of M(r) approximately 48,000. Anti-BHP14 inhibited, in a concentration-dependent manner, BH activity in rabbit
liver cytosolic fractions, as measured by deamido-BLM A2 formation. Thus, we have generated an epitope-specific neutralizing
antibody to rabbit BH, which can block the metabolism of BLM by homogenates from rabbit tissue. These results suggest that
the LAVLEQEPIVLPA epitope of rabbit BH is involved in the metabolism of BLM or is topologically near the active site. Furthermore,
a BLM-resistant squamous carcinoma (C-10E) exhibited slightly more immunoreactivity, by enzyme-linked immunosorbent assay,
to anti-BHP14 than did the parental A-253 cells, and a partially revertant (C-10E ND) cell line had intermediate anti-BHP14
binding. BH activity in these cell lines was in the same rank order as antibody binding, but differences in immunoreactivity
were less than differences in enzymatic activity. Our epitope-specific neutralizing antibody should be useful in the further
characterization of BH. |
| format | Article |
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sequence data obtained from a tryptic digest fragment of purified rabbit lung BH were used to synthesize a 14-amino acid peptide
(LAVLEQEPIVLPAK; BHP14), which was conjugated to horseshoe crab hemocyanin and used to produce rabbit antiserum that was immunoreactive
to both BHP14 and rabbit BH. Anti-BHP14 binding to BHP14 could be competitively blocked by the presence of either BHP14 or
BH. Anti-BHP14 recognized both purified rabbit liver BH and postmicrosomal fraction from rabbit liver on Western blot, as
a single band of M(r) approximately 48,000. Anti-BHP14 inhibited, in a concentration-dependent manner, BH activity in rabbit
liver cytosolic fractions, as measured by deamido-BLM A2 formation. Thus, we have generated an epitope-specific neutralizing
antibody to rabbit BH, which can block the metabolism of BLM by homogenates from rabbit tissue. These results suggest that
the LAVLEQEPIVLPA epitope of rabbit BH is involved in the metabolism of BLM or is topologically near the active site. Furthermore,
a BLM-resistant squamous carcinoma (C-10E) exhibited slightly more immunoreactivity, by enzyme-linked immunosorbent assay,
to anti-BHP14 than did the parental A-253 cells, and a partially revertant (C-10E ND) cell line had intermediate anti-BHP14
binding. BH activity in these cell lines was in the same rank order as antibody binding, but differences in immunoreactivity
were less than differences in enzymatic activity. Our epitope-specific neutralizing antibody should be useful in the further
characterization of BH.</description><identifier>ISSN: 0026-895X</identifier><identifier>EISSN: 1521-0111</identifier><identifier>PMID: 1378925</identifier><identifier>CODEN: MOPMA3</identifier><language>eng</language><publisher>Bethesda, MD: American Society for Pharmacology and Experimental Therapeutics</publisher><subject>Amino Acid Sequence ; Animals ; antibodies ; Antibodies - genetics ; Antibodies - immunology ; Antineoplastic agents ; Binding, Competitive ; Biological and medical sciences ; bleomycin hydrolase ; Blotting, Western ; Carcinoma, Squamous Cell - immunology ; Chromatography, High Pressure Liquid ; Cysteine Endopeptidases ; Epitopes - immunology ; General aspects ; Glycoside Hydrolases - antagonists & inhibitors ; Glycoside Hydrolases - immunology ; Head and Neck Neoplasms - immunology ; Humans ; immunoreactivity ; Liver - enzymology ; Lung - enzymology ; Medical sciences ; Molecular Sequence Data ; neutralization ; Pharmacology. Drug treatments ; Rabbits ; Tumor Cells, Cultured</subject><ispartof>Molecular pharmacology, 1992-07, Vol.42 (1), p.57-62</ispartof><rights>1992 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=5573392$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/1378925$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>MORRIS, G</creatorcontrib><creatorcontrib>MISTRY, J. S</creatorcontrib><creatorcontrib>JANI, J. P</creatorcontrib><creatorcontrib>MIGNANO, J. E</creatorcontrib><creatorcontrib>SEBTI, S. M</creatorcontrib><creatorcontrib>LAZO, J. S</creatorcontrib><title>Neutralization of bleomycin hydrolase by an epitope-specific antibody</title><title>Molecular pharmacology</title><addtitle>Mol Pharmacol</addtitle><description>Bleomycin hydrolase (BH) is a cysteine proteinase that terminates the pharmacological action of bleomycin (BLM). Amino acid
sequence data obtained from a tryptic digest fragment of purified rabbit lung BH were used to synthesize a 14-amino acid peptide
(LAVLEQEPIVLPAK; BHP14), which was conjugated to horseshoe crab hemocyanin and used to produce rabbit antiserum that was immunoreactive
to both BHP14 and rabbit BH. Anti-BHP14 binding to BHP14 could be competitively blocked by the presence of either BHP14 or
BH. Anti-BHP14 recognized both purified rabbit liver BH and postmicrosomal fraction from rabbit liver on Western blot, as
a single band of M(r) approximately 48,000. Anti-BHP14 inhibited, in a concentration-dependent manner, BH activity in rabbit
liver cytosolic fractions, as measured by deamido-BLM A2 formation. Thus, we have generated an epitope-specific neutralizing
antibody to rabbit BH, which can block the metabolism of BLM by homogenates from rabbit tissue. These results suggest that
the LAVLEQEPIVLPA epitope of rabbit BH is involved in the metabolism of BLM or is topologically near the active site. Furthermore,
a BLM-resistant squamous carcinoma (C-10E) exhibited slightly more immunoreactivity, by enzyme-linked immunosorbent assay,
to anti-BHP14 than did the parental A-253 cells, and a partially revertant (C-10E ND) cell line had intermediate anti-BHP14
binding. BH activity in these cell lines was in the same rank order as antibody binding, but differences in immunoreactivity
were less than differences in enzymatic activity. Our epitope-specific neutralizing antibody should be useful in the further
characterization of BH.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>antibodies</subject><subject>Antibodies - genetics</subject><subject>Antibodies - immunology</subject><subject>Antineoplastic agents</subject><subject>Binding, Competitive</subject><subject>Biological and medical sciences</subject><subject>bleomycin hydrolase</subject><subject>Blotting, Western</subject><subject>Carcinoma, Squamous Cell - immunology</subject><subject>Chromatography, High Pressure Liquid</subject><subject>Cysteine Endopeptidases</subject><subject>Epitopes - immunology</subject><subject>General aspects</subject><subject>Glycoside Hydrolases - antagonists & inhibitors</subject><subject>Glycoside Hydrolases - immunology</subject><subject>Head and Neck Neoplasms - immunology</subject><subject>Humans</subject><subject>immunoreactivity</subject><subject>Liver - enzymology</subject><subject>Lung - enzymology</subject><subject>Medical sciences</subject><subject>Molecular Sequence Data</subject><subject>neutralization</subject><subject>Pharmacology. Drug treatments</subject><subject>Rabbits</subject><subject>Tumor Cells, Cultured</subject><issn>0026-895X</issn><issn>1521-0111</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1992</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0EtLxDAUBeAgyjiO_gShC3FXyLNplzKMDxh0Mwt3Ic9pJG1q0iL11zvioEtXB-75uItzApaIYVRChNApWEKIq7Ju2Os5uMj5DUJEWQ0XYIEIrxvMlmDzbKcxyeA_5ehjX0RXqGBjN2vfF-1sUgwy20LNhewLO_gxDrbMg9XeeX24jV5FM1-CMydDtlfHXIHd_Wa3fiy3Lw9P67tt2eKGjaVCFdU1NcYiVdPKKAMrpbHWmNeMEMetItZQ1nBsnEWIY4QbiCl0kCjnyArc_rwdUnyfbB5F57O2IcjexikLTiCvWE3_hagiFEP6Da-PcFKdNWJIvpNpFsd9Dv3NsZdZy-CS7LXPv4wxTkiD_1jr9-2HT1YMrUyd1DHE_SwoFkgc6BdOdnwo</recordid><startdate>19920701</startdate><enddate>19920701</enddate><creator>MORRIS, G</creator><creator>MISTRY, J. S</creator><creator>JANI, J. P</creator><creator>MIGNANO, J. E</creator><creator>SEBTI, S. M</creator><creator>LAZO, J. S</creator><general>American Society for Pharmacology and Experimental Therapeutics</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>7QL</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>M81</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>19920701</creationdate><title>Neutralization of bleomycin hydrolase by an epitope-specific antibody</title><author>MORRIS, G ; MISTRY, J. S ; JANI, J. P ; MIGNANO, J. E ; SEBTI, S. M ; LAZO, J. S</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-h295t-b164c84dde1b846dbd06bc2cc278533f7eb3ed45972dfe11721290240f03bff3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1992</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>antibodies</topic><topic>Antibodies - genetics</topic><topic>Antibodies - immunology</topic><topic>Antineoplastic agents</topic><topic>Binding, Competitive</topic><topic>Biological and medical sciences</topic><topic>bleomycin hydrolase</topic><topic>Blotting, Western</topic><topic>Carcinoma, Squamous Cell - immunology</topic><topic>Chromatography, High Pressure Liquid</topic><topic>Cysteine Endopeptidases</topic><topic>Epitopes - immunology</topic><topic>General aspects</topic><topic>Glycoside Hydrolases - antagonists & inhibitors</topic><topic>Glycoside Hydrolases - immunology</topic><topic>Head and Neck Neoplasms - immunology</topic><topic>Humans</topic><topic>immunoreactivity</topic><topic>Liver - enzymology</topic><topic>Lung - enzymology</topic><topic>Medical sciences</topic><topic>Molecular Sequence Data</topic><topic>neutralization</topic><topic>Pharmacology. Drug treatments</topic><topic>Rabbits</topic><topic>Tumor Cells, Cultured</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>MORRIS, G</creatorcontrib><creatorcontrib>MISTRY, J. S</creatorcontrib><creatorcontrib>JANI, J. P</creatorcontrib><creatorcontrib>MIGNANO, J. E</creatorcontrib><creatorcontrib>SEBTI, S. M</creatorcontrib><creatorcontrib>LAZO, J. S</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</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>Molecular pharmacology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>MORRIS, G</au><au>MISTRY, J. S</au><au>JANI, J. P</au><au>MIGNANO, J. E</au><au>SEBTI, S. M</au><au>LAZO, J. S</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Neutralization of bleomycin hydrolase by an epitope-specific antibody</atitle><jtitle>Molecular pharmacology</jtitle><addtitle>Mol Pharmacol</addtitle><date>1992-07-01</date><risdate>1992</risdate><volume>42</volume><issue>1</issue><spage>57</spage><epage>62</epage><pages>57-62</pages><issn>0026-895X</issn><eissn>1521-0111</eissn><coden>MOPMA3</coden><abstract>Bleomycin hydrolase (BH) is a cysteine proteinase that terminates the pharmacological action of bleomycin (BLM). Amino acid
sequence data obtained from a tryptic digest fragment of purified rabbit lung BH were used to synthesize a 14-amino acid peptide
(LAVLEQEPIVLPAK; BHP14), which was conjugated to horseshoe crab hemocyanin and used to produce rabbit antiserum that was immunoreactive
to both BHP14 and rabbit BH. Anti-BHP14 binding to BHP14 could be competitively blocked by the presence of either BHP14 or
BH. Anti-BHP14 recognized both purified rabbit liver BH and postmicrosomal fraction from rabbit liver on Western blot, as
a single band of M(r) approximately 48,000. Anti-BHP14 inhibited, in a concentration-dependent manner, BH activity in rabbit
liver cytosolic fractions, as measured by deamido-BLM A2 formation. Thus, we have generated an epitope-specific neutralizing
antibody to rabbit BH, which can block the metabolism of BLM by homogenates from rabbit tissue. These results suggest that
the LAVLEQEPIVLPA epitope of rabbit BH is involved in the metabolism of BLM or is topologically near the active site. Furthermore,
a BLM-resistant squamous carcinoma (C-10E) exhibited slightly more immunoreactivity, by enzyme-linked immunosorbent assay,
to anti-BHP14 than did the parental A-253 cells, and a partially revertant (C-10E ND) cell line had intermediate anti-BHP14
binding. BH activity in these cell lines was in the same rank order as antibody binding, but differences in immunoreactivity
were less than differences in enzymatic activity. Our epitope-specific neutralizing antibody should be useful in the further
characterization of BH.</abstract><cop>Bethesda, MD</cop><pub>American Society for Pharmacology and Experimental Therapeutics</pub><pmid>1378925</pmid><tpages>6</tpages></addata></record> |
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| ispartof | Molecular pharmacology, 1992-07, Vol.42 (1), p.57-62 |
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| language | eng |
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| source | MEDLINE; EZB-FREE-00999 freely available EZB journals |
| subjects | Amino Acid Sequence Animals antibodies Antibodies - genetics Antibodies - immunology Antineoplastic agents Binding, Competitive Biological and medical sciences bleomycin hydrolase Blotting, Western Carcinoma, Squamous Cell - immunology Chromatography, High Pressure Liquid Cysteine Endopeptidases Epitopes - immunology General aspects Glycoside Hydrolases - antagonists & inhibitors Glycoside Hydrolases - immunology Head and Neck Neoplasms - immunology Humans immunoreactivity Liver - enzymology Lung - enzymology Medical sciences Molecular Sequence Data neutralization Pharmacology. Drug treatments Rabbits Tumor Cells, Cultured |
| title | Neutralization of bleomycin hydrolase by an epitope-specific antibody |
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