Differential stimulation of NAD kinase and binding of peptide substrates by wild-type and mutant plant calmodulin isoforms

Birong Liao, Margaret C. Gawienowski, Raymond E. Zielinski

Research output: Contribution to journalArticle

Abstract

Calmodulin from Arabidopsis thaliana consists of at least four isoforms, which differ in their deduced amino acid sequences by as many as six conservative substitutions. To determine whether these differences are biochemically significant, cDNAs encoding three of the four isoforms were engineered to produce recombinant proteins in Escherichia coli, purified to apparent homogeneity, and assayed for their abilities to activate pea leaf NAD kinase in vitro. The CaM-2 isoform was a significantly more efficient activator of NAD kinase compared with the CaM-4 and -6 isoforms based on both the apparent V(max) it elicited and the K0.5 activation. These results are consistent with the hypothesis that the Arabidopsis CaM isoforms have evolved to optimize the protein's interaction with different Ca2+/CaM-regulated target enzymes. The ability to activate NAD kinase was also investigated for a carboxy-terminal nonsense mutant of CaM-6 (CaM-6M), which substituted 14 hydrophilic amino acids for a region of seven amino acids that normally form an exposed hydrophobic surface when wt CaM-6 binds Ca2+. CaM-6M-activated NAD kinase displayed an apparent V(max) that was reduced 40% and a K0.5 that was an order of magnitude greater than the CaM-6-activated enzyme. The Ca2+-dependence of CaM-6M to activate NAD kinase was identical to that of CaM-6, but CaM-6M bound synthetic peptide substrates with lower apparent affinity than did CaM-6. Thus, the carboxy-terminal hydrophobic domain of CaM appears to be critical for its interaction with NAD kinase. In contrast, amino-terminal fusions of a hydrophilic, α-helical 12-residue c-myc epitope tag to CaM-2 and -4 yielded proteins that activated NAD kinase to apparent V(max) values within 10% of those obtained with the wild-type CaM proteins.

Original languageEnglish (US)
Pages (from-to)53-60
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume327
Issue number1
DOIs
StatePublished - Mar 1 1996

Fingerprint

Calmodulin
Protein Isoforms
Peptides
Substrates
Arabidopsis
Amino Acids
Proteins
Peas
Enzymes
NAD kinase
Recombinant Proteins
Escherichia coli
Epitopes
Amino Acid Sequence
Substitution reactions
Fusion reactions
Complementary DNA
Chemical activation

Keywords

  • Arabidopsis thaliana
  • calcium
  • signal transduction

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Differential stimulation of NAD kinase and binding of peptide substrates by wild-type and mutant plant calmodulin isoforms. / Liao, Birong; Gawienowski, Margaret C.; Zielinski, Raymond E.

In: Archives of Biochemistry and Biophysics, Vol. 327, No. 1, 01.03.1996, p. 53-60.

Research output: Contribution to journalArticle

Liao, Birong ; Gawienowski, Margaret C. ; Zielinski, Raymond E. / Differential stimulation of NAD kinase and binding of peptide substrates by wild-type and mutant plant calmodulin isoforms. In: Archives of Biochemistry and Biophysics. 1996 ; Vol. 327, No. 1. pp. 53-60.
@article{e771b6bc2ed14be2b8ad351fd2f8fe72,
title = "Differential stimulation of NAD kinase and binding of peptide substrates by wild-type and mutant plant calmodulin isoforms",
abstract = "Calmodulin from Arabidopsis thaliana consists of at least four isoforms, which differ in their deduced amino acid sequences by as many as six conservative substitutions. To determine whether these differences are biochemically significant, cDNAs encoding three of the four isoforms were engineered to produce recombinant proteins in Escherichia coli, purified to apparent homogeneity, and assayed for their abilities to activate pea leaf NAD kinase in vitro. The CaM-2 isoform was a significantly more efficient activator of NAD kinase compared with the CaM-4 and -6 isoforms based on both the apparent V(max) it elicited and the K0.5 activation. These results are consistent with the hypothesis that the Arabidopsis CaM isoforms have evolved to optimize the protein's interaction with different Ca2+/CaM-regulated target enzymes. The ability to activate NAD kinase was also investigated for a carboxy-terminal nonsense mutant of CaM-6 (CaM-6M), which substituted 14 hydrophilic amino acids for a region of seven amino acids that normally form an exposed hydrophobic surface when wt CaM-6 binds Ca2+. CaM-6M-activated NAD kinase displayed an apparent V(max) that was reduced 40{\%} and a K0.5 that was an order of magnitude greater than the CaM-6-activated enzyme. The Ca2+-dependence of CaM-6M to activate NAD kinase was identical to that of CaM-6, but CaM-6M bound synthetic peptide substrates with lower apparent affinity than did CaM-6. Thus, the carboxy-terminal hydrophobic domain of CaM appears to be critical for its interaction with NAD kinase. In contrast, amino-terminal fusions of a hydrophilic, α-helical 12-residue c-myc epitope tag to CaM-2 and -4 yielded proteins that activated NAD kinase to apparent V(max) values within 10{\%} of those obtained with the wild-type CaM proteins.",
keywords = "Arabidopsis thaliana, calcium, signal transduction",
author = "Birong Liao and Gawienowski, {Margaret C.} and Zielinski, {Raymond E.}",
year = "1996",
month = "3",
day = "1",
doi = "10.1006/abbi.1996.0092",
language = "English (US)",
volume = "327",
pages = "53--60",
journal = "Archives of Biochemistry and Biophysics",
issn = "0003-9861",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Differential stimulation of NAD kinase and binding of peptide substrates by wild-type and mutant plant calmodulin isoforms

AU - Liao, Birong

AU - Gawienowski, Margaret C.

AU - Zielinski, Raymond E.

PY - 1996/3/1

Y1 - 1996/3/1

N2 - Calmodulin from Arabidopsis thaliana consists of at least four isoforms, which differ in their deduced amino acid sequences by as many as six conservative substitutions. To determine whether these differences are biochemically significant, cDNAs encoding three of the four isoforms were engineered to produce recombinant proteins in Escherichia coli, purified to apparent homogeneity, and assayed for their abilities to activate pea leaf NAD kinase in vitro. The CaM-2 isoform was a significantly more efficient activator of NAD kinase compared with the CaM-4 and -6 isoforms based on both the apparent V(max) it elicited and the K0.5 activation. These results are consistent with the hypothesis that the Arabidopsis CaM isoforms have evolved to optimize the protein's interaction with different Ca2+/CaM-regulated target enzymes. The ability to activate NAD kinase was also investigated for a carboxy-terminal nonsense mutant of CaM-6 (CaM-6M), which substituted 14 hydrophilic amino acids for a region of seven amino acids that normally form an exposed hydrophobic surface when wt CaM-6 binds Ca2+. CaM-6M-activated NAD kinase displayed an apparent V(max) that was reduced 40% and a K0.5 that was an order of magnitude greater than the CaM-6-activated enzyme. The Ca2+-dependence of CaM-6M to activate NAD kinase was identical to that of CaM-6, but CaM-6M bound synthetic peptide substrates with lower apparent affinity than did CaM-6. Thus, the carboxy-terminal hydrophobic domain of CaM appears to be critical for its interaction with NAD kinase. In contrast, amino-terminal fusions of a hydrophilic, α-helical 12-residue c-myc epitope tag to CaM-2 and -4 yielded proteins that activated NAD kinase to apparent V(max) values within 10% of those obtained with the wild-type CaM proteins.

AB - Calmodulin from Arabidopsis thaliana consists of at least four isoforms, which differ in their deduced amino acid sequences by as many as six conservative substitutions. To determine whether these differences are biochemically significant, cDNAs encoding three of the four isoforms were engineered to produce recombinant proteins in Escherichia coli, purified to apparent homogeneity, and assayed for their abilities to activate pea leaf NAD kinase in vitro. The CaM-2 isoform was a significantly more efficient activator of NAD kinase compared with the CaM-4 and -6 isoforms based on both the apparent V(max) it elicited and the K0.5 activation. These results are consistent with the hypothesis that the Arabidopsis CaM isoforms have evolved to optimize the protein's interaction with different Ca2+/CaM-regulated target enzymes. The ability to activate NAD kinase was also investigated for a carboxy-terminal nonsense mutant of CaM-6 (CaM-6M), which substituted 14 hydrophilic amino acids for a region of seven amino acids that normally form an exposed hydrophobic surface when wt CaM-6 binds Ca2+. CaM-6M-activated NAD kinase displayed an apparent V(max) that was reduced 40% and a K0.5 that was an order of magnitude greater than the CaM-6-activated enzyme. The Ca2+-dependence of CaM-6M to activate NAD kinase was identical to that of CaM-6, but CaM-6M bound synthetic peptide substrates with lower apparent affinity than did CaM-6. Thus, the carboxy-terminal hydrophobic domain of CaM appears to be critical for its interaction with NAD kinase. In contrast, amino-terminal fusions of a hydrophilic, α-helical 12-residue c-myc epitope tag to CaM-2 and -4 yielded proteins that activated NAD kinase to apparent V(max) values within 10% of those obtained with the wild-type CaM proteins.

KW - Arabidopsis thaliana

KW - calcium

KW - signal transduction

UR - http://www.scopus.com/inward/record.url?scp=0030063768&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030063768&partnerID=8YFLogxK

U2 - 10.1006/abbi.1996.0092

DO - 10.1006/abbi.1996.0092

M3 - Article

C2 - 8615696

AN - SCOPUS:0030063768

VL - 327

SP - 53

EP - 60

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

IS - 1

ER -