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Evidence for a Complex Regulating the in Vivo Activities of Early Enzymes Induced by Bacteriophage T4
An in vivo assay of enzyme activity has been employed to investigate the mechanism of control of enzymes synthesizing deoxyribonucleotides after bacteriophage T4 infection. The assay is based on the release of tritium from 5-labeled pyrimidine nucleotide substrates into water. At least two phage-ind...
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| Published in: | The Journal of biological chemistry 1974-12, Vol.249 (23), p.7613-7622 |
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| container_end_page | 7622 |
| container_issue | 23 |
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| container_title | The Journal of biological chemistry |
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| creator | Tomich, P K Chiu, C S Wovcha, M G Greenberg, G R |
| description | An in vivo assay of enzyme activity has been employed to investigate the mechanism of control of enzymes synthesizing deoxyribonucleotides
after bacteriophage T4 infection. The assay is based on the release of tritium from 5-labeled pyrimidine nucleotide substrates
into water.
At least two phage-induced early enzymes, deoxycytidylate hydroxymethylase and thymidylate synthetase, which are synthesized
within a few minutes after infection as measured in extracts, do not function in vivo immediately after their formation. Instead, in vivo these enzyme activities initiate about 5 min after infection at 30°. The activities initially increase exponentially, and
then become linear. This exponential activation process does not require concomitant protein synthesis. A number of experiments
rule against substrate limitation, feedback, or isotope dilution as an explanation of these kinetic data. Thus, the same kinetic
behavior in vivo is observed on infection by mutants which are unable to synthesize T4 DNA (DO) and accumulate deoxyribonucleotides. However,
amber mutants of gene 43, the structural gene for T4-induced DNA polymerase, are exceptions. Infection by these mutants clearly
showed a reduced initial rate of 3 H release.
Concurrent measurement of DNA synthesis and of in vivo enzyme activity indicates that these two processes follow the same kinetics and coincide in time. These results suggest that
the limiting factor in DNA replication is the rate of formation of the deoxyribonucleotide substrates. From these and previous
studies we have postulated that dCMP hydroxymethylase and thymidylate synthetase and apparently other enzymes forming deoxyribonucleotides
must, to be active in vivo , become part of a complex. We propose that the exponential activation process represents the formation of this complex from
its components. |
| doi_str_mv | 10.1016/S0021-9258(19)81282-6 |
| format | article |
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after bacteriophage T4 infection. The assay is based on the release of tritium from 5-labeled pyrimidine nucleotide substrates
into water.
At least two phage-induced early enzymes, deoxycytidylate hydroxymethylase and thymidylate synthetase, which are synthesized
within a few minutes after infection as measured in extracts, do not function in vivo immediately after their formation. Instead, in vivo these enzyme activities initiate about 5 min after infection at 30°. The activities initially increase exponentially, and
then become linear. This exponential activation process does not require concomitant protein synthesis. A number of experiments
rule against substrate limitation, feedback, or isotope dilution as an explanation of these kinetic data. Thus, the same kinetic
behavior in vivo is observed on infection by mutants which are unable to synthesize T4 DNA (DO) and accumulate deoxyribonucleotides. However,
amber mutants of gene 43, the structural gene for T4-induced DNA polymerase, are exceptions. Infection by these mutants clearly
showed a reduced initial rate of 3 H release.
Concurrent measurement of DNA synthesis and of in vivo enzyme activity indicates that these two processes follow the same kinetics and coincide in time. These results suggest that
the limiting factor in DNA replication is the rate of formation of the deoxyribonucleotide substrates. From these and previous
studies we have postulated that dCMP hydroxymethylase and thymidylate synthetase and apparently other enzymes forming deoxyribonucleotides
must, to be active in vivo , become part of a complex. We propose that the exponential activation process represents the formation of this complex from
its components.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1016/S0021-9258(19)81282-6</identifier><identifier>PMID: 4612038</identifier><language>eng</language><publisher>United States: American Society for Biochemistry and Molecular Biology</publisher><subject>Chromatography, Ion Exchange ; Coliphages - enzymology ; Deoxycytidine Monophosphate ; DNA Nucleotidyltransferases - metabolism ; DNA Viruses - metabolism ; DNA, Bacterial - biosynthesis ; DNA, Viral - biosynthesis ; Escherichia coli - enzymology ; Genes ; Kinetics ; Macromolecular Substances ; Methyltransferases - metabolism ; Mutation ; Phenotype ; Ribonucleoside Diphosphate Reductase - metabolism ; RNA, Bacterial - biosynthesis ; Thymidylate Synthase - metabolism ; Time Factors ; Transcription, Genetic ; Transferases - metabolism ; Tritium ; Uridine - metabolism ; Virus Replication</subject><ispartof>The Journal of biological chemistry, 1974-12, Vol.249 (23), p.7613-7622</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c2946-b2fc794c6a8c9e497a30d5658e30bba6acb4ff5219850cb898259af43b02a1d93</citedby><cites>FETCH-LOGICAL-c2946-b2fc794c6a8c9e497a30d5658e30bba6acb4ff5219850cb898259af43b02a1d93</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>315,786,790,27957,27958</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/4612038$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Tomich, P K</creatorcontrib><creatorcontrib>Chiu, C S</creatorcontrib><creatorcontrib>Wovcha, M G</creatorcontrib><creatorcontrib>Greenberg, G R</creatorcontrib><title>Evidence for a Complex Regulating the in Vivo Activities of Early Enzymes Induced by Bacteriophage T4</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>An in vivo assay of enzyme activity has been employed to investigate the mechanism of control of enzymes synthesizing deoxyribonucleotides
after bacteriophage T4 infection. The assay is based on the release of tritium from 5-labeled pyrimidine nucleotide substrates
into water.
At least two phage-induced early enzymes, deoxycytidylate hydroxymethylase and thymidylate synthetase, which are synthesized
within a few minutes after infection as measured in extracts, do not function in vivo immediately after their formation. Instead, in vivo these enzyme activities initiate about 5 min after infection at 30°. The activities initially increase exponentially, and
then become linear. This exponential activation process does not require concomitant protein synthesis. A number of experiments
rule against substrate limitation, feedback, or isotope dilution as an explanation of these kinetic data. Thus, the same kinetic
behavior in vivo is observed on infection by mutants which are unable to synthesize T4 DNA (DO) and accumulate deoxyribonucleotides. However,
amber mutants of gene 43, the structural gene for T4-induced DNA polymerase, are exceptions. Infection by these mutants clearly
showed a reduced initial rate of 3 H release.
Concurrent measurement of DNA synthesis and of in vivo enzyme activity indicates that these two processes follow the same kinetics and coincide in time. These results suggest that
the limiting factor in DNA replication is the rate of formation of the deoxyribonucleotide substrates. From these and previous
studies we have postulated that dCMP hydroxymethylase and thymidylate synthetase and apparently other enzymes forming deoxyribonucleotides
must, to be active in vivo , become part of a complex. We propose that the exponential activation process represents the formation of this complex from
its components.</description><subject>Chromatography, Ion Exchange</subject><subject>Coliphages - enzymology</subject><subject>Deoxycytidine Monophosphate</subject><subject>DNA Nucleotidyltransferases - metabolism</subject><subject>DNA Viruses - metabolism</subject><subject>DNA, Bacterial - biosynthesis</subject><subject>DNA, Viral - biosynthesis</subject><subject>Escherichia coli - enzymology</subject><subject>Genes</subject><subject>Kinetics</subject><subject>Macromolecular Substances</subject><subject>Methyltransferases - metabolism</subject><subject>Mutation</subject><subject>Phenotype</subject><subject>Ribonucleoside Diphosphate Reductase - metabolism</subject><subject>RNA, Bacterial - biosynthesis</subject><subject>Thymidylate Synthase - metabolism</subject><subject>Time Factors</subject><subject>Transcription, Genetic</subject><subject>Transferases - metabolism</subject><subject>Tritium</subject><subject>Uridine - metabolism</subject><subject>Virus Replication</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1974</creationdate><recordtype>article</recordtype><recordid>eNo9kFtr3DAQRkVoSTeXnxAQBEr64EY3a6XHZNmmgUChTULehCSP1yq2tZHsTTe_vk52ybwMzHdmBg5CZ5R8p4TKyz-EMFpoVqoLqr8pyhQr5AGaUaJ4wUv69AnNPpAv6Cjnv2QqoekhOhSSMsLVDMFyEyroPeA6JmzxInbrFv7h37AaWzuEfoWHBnDo8WPYRHzlh7AJQ4CMY42XNrVbvOxft900uO2r0UOF3RZfWz9ACnHd2BXge3GCPte2zXC678fo4cfyfvGzuPt1c7u4uis800IWjtV-roWXVnkNQs8tJ1UpSwWcOGel9U7UdcmoViXxTmnFSm1rwR1hllaaH6Ovu7vrFJ9HyIPpQvbQtraHOGYz8YoTLSaw3IE-xZwT1GadQmfT1lBi3vSad73mzZ2h2rzrNXLaO9s_GF0H1cfW3ueUn-_yJqyal5DAuBB9A51hQhvGzVxSzv8DeieA_g</recordid><startdate>19741210</startdate><enddate>19741210</enddate><creator>Tomich, P K</creator><creator>Chiu, C S</creator><creator>Wovcha, M G</creator><creator>Greenberg, G R</creator><general>American Society for Biochemistry and Molecular Biology</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>19741210</creationdate><title>Evidence for a Complex Regulating the in Vivo Activities of Early Enzymes Induced by Bacteriophage T4</title><author>Tomich, P K ; Chiu, C S ; Wovcha, M G ; Greenberg, G R</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c2946-b2fc794c6a8c9e497a30d5658e30bba6acb4ff5219850cb898259af43b02a1d93</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1974</creationdate><topic>Chromatography, Ion Exchange</topic><topic>Coliphages - enzymology</topic><topic>Deoxycytidine Monophosphate</topic><topic>DNA Nucleotidyltransferases - metabolism</topic><topic>DNA Viruses - metabolism</topic><topic>DNA, Bacterial - biosynthesis</topic><topic>DNA, Viral - biosynthesis</topic><topic>Escherichia coli - enzymology</topic><topic>Genes</topic><topic>Kinetics</topic><topic>Macromolecular Substances</topic><topic>Methyltransferases - metabolism</topic><topic>Mutation</topic><topic>Phenotype</topic><topic>Ribonucleoside Diphosphate Reductase - metabolism</topic><topic>RNA, Bacterial - biosynthesis</topic><topic>Thymidylate Synthase - metabolism</topic><topic>Time Factors</topic><topic>Transcription, Genetic</topic><topic>Transferases - metabolism</topic><topic>Tritium</topic><topic>Uridine - metabolism</topic><topic>Virus Replication</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Tomich, P K</creatorcontrib><creatorcontrib>Chiu, C S</creatorcontrib><creatorcontrib>Wovcha, M G</creatorcontrib><creatorcontrib>Greenberg, G R</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Tomich, P K</au><au>Chiu, C S</au><au>Wovcha, M G</au><au>Greenberg, G R</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Evidence for a Complex Regulating the in Vivo Activities of Early Enzymes Induced by Bacteriophage T4</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1974-12-10</date><risdate>1974</risdate><volume>249</volume><issue>23</issue><spage>7613</spage><epage>7622</epage><pages>7613-7622</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><notes>ObjectType-Article-1</notes><notes>SourceType-Scholarly Journals-1</notes><notes>ObjectType-Feature-2</notes><notes>content type line 23</notes><abstract>An in vivo assay of enzyme activity has been employed to investigate the mechanism of control of enzymes synthesizing deoxyribonucleotides
after bacteriophage T4 infection. The assay is based on the release of tritium from 5-labeled pyrimidine nucleotide substrates
into water.
At least two phage-induced early enzymes, deoxycytidylate hydroxymethylase and thymidylate synthetase, which are synthesized
within a few minutes after infection as measured in extracts, do not function in vivo immediately after their formation. Instead, in vivo these enzyme activities initiate about 5 min after infection at 30°. The activities initially increase exponentially, and
then become linear. This exponential activation process does not require concomitant protein synthesis. A number of experiments
rule against substrate limitation, feedback, or isotope dilution as an explanation of these kinetic data. Thus, the same kinetic
behavior in vivo is observed on infection by mutants which are unable to synthesize T4 DNA (DO) and accumulate deoxyribonucleotides. However,
amber mutants of gene 43, the structural gene for T4-induced DNA polymerase, are exceptions. Infection by these mutants clearly
showed a reduced initial rate of 3 H release.
Concurrent measurement of DNA synthesis and of in vivo enzyme activity indicates that these two processes follow the same kinetics and coincide in time. These results suggest that
the limiting factor in DNA replication is the rate of formation of the deoxyribonucleotide substrates. From these and previous
studies we have postulated that dCMP hydroxymethylase and thymidylate synthetase and apparently other enzymes forming deoxyribonucleotides
must, to be active in vivo , become part of a complex. We propose that the exponential activation process represents the formation of this complex from
its components.</abstract><cop>United States</cop><pub>American Society for Biochemistry and Molecular Biology</pub><pmid>4612038</pmid><doi>10.1016/S0021-9258(19)81282-6</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | The Journal of biological chemistry, 1974-12, Vol.249 (23), p.7613-7622 |
| issn | 0021-9258 1083-351X |
| language | eng |
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| source | BACON - Elsevier - GLOBAL_SCIENCEDIRECT-OPENACCESS |
| subjects | Chromatography, Ion Exchange Coliphages - enzymology Deoxycytidine Monophosphate DNA Nucleotidyltransferases - metabolism DNA Viruses - metabolism DNA, Bacterial - biosynthesis DNA, Viral - biosynthesis Escherichia coli - enzymology Genes Kinetics Macromolecular Substances Methyltransferases - metabolism Mutation Phenotype Ribonucleoside Diphosphate Reductase - metabolism RNA, Bacterial - biosynthesis Thymidylate Synthase - metabolism Time Factors Transcription, Genetic Transferases - metabolism Tritium Uridine - metabolism Virus Replication |
| title | Evidence for a Complex Regulating the in Vivo Activities of Early Enzymes Induced by Bacteriophage T4 |
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