Pectinmethylesterase isoforms from Vigna radiata hypocotyl cell walls: kinetic properties and molecular cloning of a cDNA encoding the most alkaline isoform.
Peptide maps and partial amino acid sequences of the 3 main pectinmethylesterases (PMEs) solubilized from mung bean hypocotyl cell walls demonstrated that these proteins were different isozymes originating from a small multigene family. A cDNA clone encoding the most alkaline PME (PE gamma) have been obtained by PCR using degenerate oligonucleotide primers. Combining the protein and nucleotide sequencing data, the complete amino acid sequence of PE gamma was determined. The nature protein is composed of 318 amino acids with a calculated Mtau of 34 677 and an estimated pI of 9.84 consistent with the values previously obtained by SDS-PAGE and IEF. It shares most of the conserved regions of previously known PMEs. Enzymatic activities of the three isoforms were differently affected by the presence of cations in the incubation medium but, in all cases, infra-optimal cation concentrations induced two opposite effects: a decrease in the Vmax and an increase in the affinity of the enzymes for their substrate. The presence of cations in the assay modulates both the number of enzyme molecules available to the demethylation reaction and the conformation of the pectin and, in turn, the affinity of the PMEs for their substrate.
Bordenave
M
M
Institut Jacques Monod, Paris, France.
Breton
C
C
Goldberg
R
R
Huet
J C
JC
Perez
S
S
Pernollet
J C
JC
D000595:Amino Acid Sequence; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D002473:Cell Wall / Q000201:enzymology*; D003001:Cloning, Molecular; D007887:Fabaceae / Q000201:enzymology / Q000235:genetics*; D006868:Hydrolysis; D018546:Hypocotyl / Q000201:enzymology*; D007527:Isoenzymes / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010368:Pectins / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010946:Plants, Medicinal; D012492:Salts / Q000494:pharmacology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D007527:Isoenzymes; D010446:Peptide Fragments; D012492:Salts; D010368:Pectins; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase