{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/20627"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/20627","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Molecular characterization of phosphoribulokinase and rubisco activase from Chlamydomonas reinhardtii","abstract":"Chlamydomonas reinhardtii phosphoribulokinase was purified to apparent homogeneity and polyclonal antibodies were prepared. Immunoblot analysis indicated antigenic similarity and a similar subunit size for the enzyme from five higher plant species and Chlamydomonas. Immunoblots also revealed substantial amounts of phosphoribulokinase protein in Chalmydomonas mutant strain 12-2B, but none in mutant strain F-60. Chlamydomonas and spinach cDNA clones containing the entire phosphoribulokinase coding region were isolated and sequenced. The Chlamydomonas and spinach mature proteins possessed 75% amino acid sequence identity and similar kinetic properties. The transit peptides possessed almost no amino acid sequence identity. Genomic DNA blot analysis suggested the presence of a single Chlamydomonas phosphoribulokinase gene. The pH optimum of the wild-type Chlamydomonas enzyme was 8.0, while that of the 12-2B enzyme was 6.5. The mutant kinase possessed a Km (Ru5P) value nearly 1000-fold greater than the wild-type value of 56 $\\mu$M. Similar Km (ATP) values of less than 100 $\\mu$M were observed with both wild-type and mutant enzymes. The wild-type Vmax was 450 $\\mu$mol min$\\sp{-1}$ mg$\\sp{-1}$, while values for the 12-2B enzyme were 140 $\\mu$mol min$\\sp{-1}$ mg$\\sp{-1}$ at pH 6.5 and 36 $\\mu$mol min$\\sp{-1}$ mg$\\sp{-1}$ at pH 7.8. Thermal stabilities of the wild-type and mutant kinases were similar. Sequence analysis of the 12-2B phosphoribulokinase gene revealed a C to T transition which created an arginine to cysteine change at position 64 of mature phosphoribulokinase. This arginine residue is conserved in the enzyme of vascular plants, algae, and photosynthetic bacteria and appears to function in binding the phosphoribulose substrate. Immunoblots and DNA sequence analysis indicated the presence of a single Chlamydomonas rubisco activase polypeptide which possessed 60 to 65% amino acid sequence identity with the higher plant polypeptides. Chlamydomonas rubisco was less effectively activated by spinach rubisco activase than was spinach rubisco. Genomic DNA blot analysis suggested the presence of a single copy Chlamydomonas rubisco activase gene.","abstract_html":"Chlamydomonas reinhardtii phosphoribulokinase was purified to apparent homogeneity and polyclonal antibodies were prepared. Immunoblot analysis indicated antigenic similarity and a similar subunit size for the enzyme from five higher plant species and Chlamydomonas. Immunoblots also revealed substantial amounts of phosphoribulokinase protein in Chalmydomonas mutant strain 12-2B, but none in mutant strain F-60. Chlamydomonas and spinach cDNA clones containing the entire phosphoribulokinase coding region were isolated and sequenced. The Chlamydomonas and spinach mature proteins possessed 75% amino acid sequence identity and similar kinetic properties. The transit peptides possessed almost no amino acid sequence identity. Genomic DNA blot analysis suggested the presence of a single Chlamydomonas phosphoribulokinase gene. The pH optimum of the wild-type Chlamydomonas enzyme was 8.0, while that of the 12-2B enzyme was 6.5. The mutant kinase possessed a Km (Ru5P) value nearly 1000-fold greater than the wild-type value of 56 <span class=\"etd-inline-math\">&mu;</span>M. Similar Km (ATP) values of less than 100 <span class=\"etd-inline-math\">&mu;</span>M were observed with both wild-type and mutant enzymes. The wild-type Vmax was 450 <span class=\"etd-inline-math\">&mu;</span>mol min$\\sp{-1}$ mg$\\sp{-1}$, while values for the 12-2B enzyme were 140 <span class=\"etd-inline-math\">&mu;</span>mol min$\\sp{-1}$ mg$\\sp{-1}$ at pH 6.5 and 36 <span class=\"etd-inline-math\">&mu;</span>mol min$\\sp{-1}$ mg$\\sp{-1}$ at pH 7.8. Thermal stabilities of the wild-type and mutant kinases were similar. Sequence analysis of the 12-2B phosphoribulokinase gene revealed a C to T transition which created an arginine to cysteine change at position 64 of mature phosphoribulokinase. This arginine residue is conserved in the enzyme of vascular plants, algae, and photosynthetic bacteria and appears to function in binding the phosphoribulose substrate. Immunoblots and DNA sequence analysis indicated the presence of a single Chlamydomonas rubisco activase polypeptide which possessed 60 to 65% amino acid sequence identity with the higher plant polypeptides. Chlamydomonas rubisco was less effectively activated by spinach rubisco activase than was spinach rubisco. Genomic DNA blot analysis suggested the presence of a single copy Chlamydomonas rubisco activase gene.","abstract_has_math":true,"creators":["Roesler, Keith Robert"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biology, Botany","degree_department":null,"school":null,"contributors":["Ogren, William L."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2011,"date_issued":"2011-05-07T12:44:41Z","date_published":"2011-05-07T12:44:41Z","updated_at":"2026-07-22T22:25:16Z","subjects":["Agriculture, Agronomy","Biology, Botany","Biology, Plant Physiology"],"languages":["eng"],"rights":["Copyright 1990 Roesler, Keith Robert"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["AAI9114390","(UMI)AAI9114390"],"render_values":[{"text":"AAI9114390","href":null,"code":true},{"text":"(UMI)AAI9114390","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2142/20627","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Ogren, William L."]},{"key":"dc:creator","label":"Author","values":["Roesler, Keith Robert"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2011-05-07T12:44:41Z","10000-01-01","1990"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biology, Botany","Crop Sciences"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Agriculture, Agronomy","Biology, Botany","Biology, Plant Physiology"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 1990 Roesler, Keith Robert"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["AAI9114390","(UMI)AAI9114390","http://hdl.handle.net/2142/20627"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Chlamydomonas reinhardtii phosphoribulokinase was purified to apparent homogeneity and polyclonal antibodies were prepared. Immunoblot analysis indicated antigenic similarity and a similar subunit size for the enzyme from five higher plant species and Chlamydomonas. Immunoblots also revealed substantial amounts of phosphoribulokinase protein in Chalmydomonas mutant strain 12-2B, but none in mutant strain F-60. Chlamydomonas and spinach cDNA clones containing the entire phosphoribulokinase coding region were isolated and sequenced. The Chlamydomonas and spinach mature proteins possessed 75% amino acid sequence identity and similar kinetic properties. The transit peptides possessed almost no amino acid sequence identity. Genomic DNA blot analysis suggested the presence of a single Chlamydomonas phosphoribulokinase gene. The pH optimum of the wild-type Chlamydomonas enzyme was 8.0, while that of the 12-2B enzyme was 6.5. The mutant kinase possessed a Km (Ru5P) value nearly 1000-fold greater than the wild-type value of 56 $\\mu$M. Similar Km (ATP) values of less than 100 $\\mu$M were observed with both wild-type and mutant enzymes. The wild-type Vmax was 450 $\\mu$mol min$\\sp{-1}$ mg$\\sp{-1}$, while values for the 12-2B enzyme were 140 $\\mu$mol min$\\sp{-1}$ mg$\\sp{-1}$ at pH 6.5 and 36 $\\mu$mol min$\\sp{-1}$ mg$\\sp{-1}$ at pH 7.8. Thermal stabilities of the wild-type and mutant kinases were similar. Sequence analysis of the 12-2B phosphoribulokinase gene revealed a C to T transition which created an arginine to cysteine change at position 64 of mature phosphoribulokinase. This arginine residue is conserved in the enzyme of vascular plants, algae, and photosynthetic bacteria and appears to function in binding the phosphoribulose substrate. Immunoblots and DNA sequence analysis indicated the presence of a single Chlamydomonas rubisco activase polypeptide which possessed 60 to 65% amino acid sequence identity with the higher plant polypeptides. Chlamydomonas rubisco was less effectively activated by spinach rubisco activase than was spinach rubisco. Genomic DNA blot analysis suggested the presence of a single copy Chlamydomonas rubisco activase gene.","Made available in DSpace on 2011-05-07T12:44:41Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 9114390.pdf: 2552398 bytes, checksum: 79cb8ba2584e9b27edd1dae07a715f6e (MD5) Previous issue date: 1990","Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T14:45:10Z Item is restricted indefinitely.","Restriction data tranferred 2014-07-01T11:19:58-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: ETDs are only available to UIUC Users without author permission","ETDs are only available to UIUC Users without author permission","U of I Only"]},{"key":"dc:title","label":"Title","values":["Molecular characterization of phosphoribulokinase and rubisco activase from Chlamydomonas reinhardtii"]}]}],"canonical_facts":{"dc:contributor":["Ogren, William L."],"dc:creator":["Roesler, Keith Robert"],"dc:date":["2011-05-07T12:44:41Z","10000-01-01","1990"],"dc:description":["Chlamydomonas reinhardtii phosphoribulokinase was purified to apparent homogeneity and polyclonal antibodies were prepared. Immunoblot analysis indicated antigenic similarity and a similar subunit size for the enzyme from five higher plant species and Chlamydomonas. Immunoblots also revealed substantial amounts of phosphoribulokinase protein in Chalmydomonas mutant strain 12-2B, but none in mutant strain F-60. Chlamydomonas and spinach cDNA clones containing the entire phosphoribulokinase coding region were isolated and sequenced. The Chlamydomonas and spinach mature proteins possessed 75% amino acid sequence identity and similar kinetic properties. The transit peptides possessed almost no amino acid sequence identity. Genomic DNA blot analysis suggested the presence of a single Chlamydomonas phosphoribulokinase gene. The pH optimum of the wild-type Chlamydomonas enzyme was 8.0, while that of the 12-2B enzyme was 6.5. The mutant kinase possessed a Km (Ru5P) value nearly 1000-fold greater than the wild-type value of 56 $\\mu$M. Similar Km (ATP) values of less than 100 $\\mu$M were observed with both wild-type and mutant enzymes. The wild-type Vmax was 450 $\\mu$mol min$\\sp{-1}$ mg$\\sp{-1}$, while values for the 12-2B enzyme were 140 $\\mu$mol min$\\sp{-1}$ mg$\\sp{-1}$ at pH 6.5 and 36 $\\mu$mol min$\\sp{-1}$ mg$\\sp{-1}$ at pH 7.8. Thermal stabilities of the wild-type and mutant kinases were similar. Sequence analysis of the 12-2B phosphoribulokinase gene revealed a C to T transition which created an arginine to cysteine change at position 64 of mature phosphoribulokinase. This arginine residue is conserved in the enzyme of vascular plants, algae, and photosynthetic bacteria and appears to function in binding the phosphoribulose substrate. Immunoblots and DNA sequence analysis indicated the presence of a single Chlamydomonas rubisco activase polypeptide which possessed 60 to 65% amino acid sequence identity with the higher plant polypeptides. Chlamydomonas rubisco was less effectively activated by spinach rubisco activase than was spinach rubisco. Genomic DNA blot analysis suggested the presence of a single copy Chlamydomonas rubisco activase gene.","Made available in DSpace on 2011-05-07T12:44:41Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 9114390.pdf: 2552398 bytes, checksum: 79cb8ba2584e9b27edd1dae07a715f6e (MD5) Previous issue date: 1990","Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T14:45:10Z Item is restricted indefinitely.","Restriction data tranferred 2014-07-01T11:19:58-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: ETDs are only available to UIUC Users without author permission","ETDs are only available to UIUC Users without author permission","U of I Only"],"dc:identifier":["AAI9114390","(UMI)AAI9114390","http://hdl.handle.net/2142/20627"],"dc:language":["eng"],"dc:rights":["Copyright 1990 Roesler, Keith Robert"],"dc:subject":["Agriculture, Agronomy","Biology, Botany","Biology, Plant Physiology"],"dc:title":["Molecular characterization of phosphoribulokinase and rubisco activase from Chlamydomonas reinhardtii"],"dc:type":["text"],"thesis:degree_discipline":["Biology, Botany","Crop Sciences"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:25:16Z"}