Biochemical, developmental, molecular, and phylogenetic studies on insect pyruvate carboxylases.

Persistent Link:
http://hdl.handle.net/10150/186864
Title:
Biochemical, developmental, molecular, and phylogenetic studies on insect pyruvate carboxylases.
Author:
Tu, Zhijian.
Issue Date:
1994
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Pyruvate carboxylase (Pyruvate: carbon dioxide ligase (ADP-forming), EC 6.4.1.1), is a biotin-dependent enzyme catalyzing a two step reaction ligating the carboxyl group to pyruvate. This enzyme was shown to be present in several tissues of ten different species of insects. Pyruvate carboxylase was purified from the thoraces of honeybee, Apis mellifera, and was found to be a tetramer consisting of 128 kDa subunits. The activity of this enzyme required acetyl-CoA, ATP, and Mg²⁺. The Kms of the enzyme for bicarbonate and pyruvate were similar to pyruvate carboxylases from other organisms. Pyruvate carboxylase was also purified from the yellow fever mosquito, Aedes aegypti. Two polypeptides of similar molecular weight (133 kDa and 128 kDa) were present and the N-terminal sequences of both were determined. The relative amounts of the 133 kDa and 128 kDa polypeptides were shown to differ in various tissues. The enzyme was found in all tissues examined and was concentrated in the thorax where the amount of the enzyme increased shortly after pupation. Pyruvate carboxylase in thoracic muscles of insects is likely to have an anaplerotic role. Pyruvate carboxylase was also found to be at high levels in the fat body preparations. Clones covering the complete cDNA of pyruvate carboxylase of Aedes aegypti were obtained. The 3942 bp nucleotide sequence including a 3585 bp coding region, was determined from these cDNA clones. The deduced 1195 amino acid sequence has a calculated Mr of 132, 200. Following a putative mitochondrial targeting sequence, three functional domains were identified including biotin carboxylase (BC), carboxyltransferase (CT), and biotin carboxyl carrier protein (BCCP). The mosquito pyruvate carboxylase amino acid sequence showed 55-75% identity to enzymes from other sources. The evolutionary relationship of pyruvate carboxylases among different organisms was analyzed. The evolution of domain structures of the biotin-dependent carboxylases including pyruvate carboxylase was also investigated. The above analysis provided evidence for the coevolution of BC and BCCP domains and early gene duplication events that shaped the family of biotin-dependent carboxylases.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Molecular biology.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Entomology; Graduate College
Degree Grantor:
University of Arizona
Committee Chair:
Hagedorn, Henry H.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleBiochemical, developmental, molecular, and phylogenetic studies on insect pyruvate carboxylases.en_US
dc.creatorTu, Zhijian.en_US
dc.contributor.authorTu, Zhijian.en_US
dc.date.issued1994en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractPyruvate carboxylase (Pyruvate: carbon dioxide ligase (ADP-forming), EC 6.4.1.1), is a biotin-dependent enzyme catalyzing a two step reaction ligating the carboxyl group to pyruvate. This enzyme was shown to be present in several tissues of ten different species of insects. Pyruvate carboxylase was purified from the thoraces of honeybee, Apis mellifera, and was found to be a tetramer consisting of 128 kDa subunits. The activity of this enzyme required acetyl-CoA, ATP, and Mg²⁺. The Kms of the enzyme for bicarbonate and pyruvate were similar to pyruvate carboxylases from other organisms. Pyruvate carboxylase was also purified from the yellow fever mosquito, Aedes aegypti. Two polypeptides of similar molecular weight (133 kDa and 128 kDa) were present and the N-terminal sequences of both were determined. The relative amounts of the 133 kDa and 128 kDa polypeptides were shown to differ in various tissues. The enzyme was found in all tissues examined and was concentrated in the thorax where the amount of the enzyme increased shortly after pupation. Pyruvate carboxylase in thoracic muscles of insects is likely to have an anaplerotic role. Pyruvate carboxylase was also found to be at high levels in the fat body preparations. Clones covering the complete cDNA of pyruvate carboxylase of Aedes aegypti were obtained. The 3942 bp nucleotide sequence including a 3585 bp coding region, was determined from these cDNA clones. The deduced 1195 amino acid sequence has a calculated Mr of 132, 200. Following a putative mitochondrial targeting sequence, three functional domains were identified including biotin carboxylase (BC), carboxyltransferase (CT), and biotin carboxyl carrier protein (BCCP). The mosquito pyruvate carboxylase amino acid sequence showed 55-75% identity to enzymes from other sources. The evolutionary relationship of pyruvate carboxylases among different organisms was analyzed. The evolution of domain structures of the biotin-dependent carboxylases including pyruvate carboxylase was also investigated. The above analysis provided evidence for the coevolution of BC and BCCP domains and early gene duplication events that shaped the family of biotin-dependent carboxylases.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectMolecular biology.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineEntomologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.chairHagedorn, Henry H.en_US
dc.contributor.committeememberChapman, Reginald F.en_US
dc.contributor.committeememberByrne, David N.en_US
dc.contributor.committeememberFeyereisen, Reneen_US
dc.contributor.committeememberWells, Michael A.en_US
dc.contributor.committeememberLaw, John H.en_US
dc.identifier.proquest9506995en_US
dc.identifier.oclc702669949en_US
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