Abstract
The primary sequence of maize 2,3-bisphosphoglycerate-independent phosphoglycerate mutase was deduced from cDNAs isolated from maize cDNA libraries by screening with specific antibodies to the cofactor-independent enzyme and from a maize genomic clone. The genomic clone provided the 5'-nucleotide sequence encoding the N-terminal amino acids which could not be obtained from the cDNA. Confirmation that the nucleotide sequence was for the cofactor-independent phosphoglycerate mutase was obtained by sequencing the peptides generated from cyanogen bromide cleavage of the purified protein. This is the first report of the amino acid sequence of a 2,3-bisphosphoglycerate cofactor-independent phosphoglycerate mutase, which consists of 559 amino acids and is twice the molecular size of the mammalian cofactor-dependent enzyme subunit. Analysis of the cofactor-independent phosphoglycerate mutase amino acid sequence revealed no identity with the cofactor-dependent mutase types. Northern blot analysis confirmed this difference since the maize cofactor-independent phosphoglycerate mutase cDNA did not hybridize with mRNA of the cofactor-dependent mutase. The lack of amino acid identity between cofactor-dependent and -independent enzymes is consistent with their different catalytic mechanisms and suggests that both enzymes are unrelated evolutionarily and arose from two independent ancestral genes. However, a constellation of residues which are involved in metal ion binding in various alkaline phosphatases is conserved in the maize cofactor-independent phosphoglycerate mutase, which suggests that the enzyme is a member of the alkaline phosphatase family of enzymes.
Highlights
The primary sequence of maize 2,3-bisphosphoglycerate-independent phosphoglycerate mutase was deduced fromcDNAs isolated from maizceDNA libraries by screening with specific antibodies to the cofactorindependent enzyme and froma maize genomic clone
The peptides generated from cyanogen bromide cleavage of the purified protein. This is the first report of the amino acid sequenceof a 2,3-bisphosphoglycerate cofactor-independent phosphoglycerate mutase, which types differ in their structure, reaction mechanism and kinetic properties
The filters were incubated with peroxidase-conjugated swine anti-rabbit IgG and developed with diaminobenzidine by the method of Davis [17].A second maize cDNA library in Xgtll was screened with random-primed labeled clones isolated from the X-ZAP screening
Summary
Materials-Enzymes were from Boehringer Mannheim, Pharmacia, and New England Biolabs. Radioactive materials were purchased from Amersham. Protein Analysis and Sequencing-Maize PGAM (E41) was purified as described previously [12]. The filters were incubated with peroxidase-conjugated swine anti-rabbit IgG and developed with diaminobenzidine by the method of Davis [17].A second maize cDNA library in Xgtll was screened with random-primed labeled clones isolated from the X-ZAP screening. Primer Extension and SI Nuclease Mapping-Poly(A) RNA was isolated from maize embryos as described previously [12], using oligo(dT)-cellulose affinity chromatography. S1 nuclease mapping was performed as described by Ausubel et al [20], using the double-stranded genomic PGAM-gl clone as template and [ T - ~ ~ P I A TenPd-labeled oligonucleotide 3 as primer. The filters were treated under the same conditions used in the Northern blotting experiments except that they were washed with 0.2 x SSC for120 min at 42 "C (four changes)
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