The calcium requirement for stability and enzymatic activity of two isoforms of barley aleurone alpha-amylase.
alpha-Amylases (EC 3.2.1.1) secreted by the aleurone layer of barley grains are Ca2+-containing metalloenzymes. We studied the effect of Ca2+ on the activity and structure of the two major groups of aleurone alpha-amylase by incubating affinity purified enzyme in solutions containing Ca2+ from pCa 4 to 7. Both groups of isoforms required one atom of Ca2+/molecule of enzyme as determined by isotope exchange, but the two groups differed by more than 10-fold in their affinity for Ca2+. Both groups of alpha-amylase were irreversibly inactivated by incubation in low Ca2+ (pCa 7). This inactivation was not due to changes in primary structure, as measured by molecular weight, but appeared to be the result of changes in secondary and tertiary structure as indicated by circular dichroism spectra, serology, lability in the presence of protease, and fluorescence spectra. Analysis of the predicted secondary structure of barley aleurone alpha-amylase indicates that the Ca2+-binding region of barley amylases is structurally similar to that of mammalian alpha-amylases. Our data indicate that micromolar levels of Ca2+ are required to stabilize the structure of barley alpha-amylases in the endoplasmic reticulum of the aleurone layer where these enzymes are synthesized.
Bush
D S
DS
Department of Plant Biology, University of California, Berkeley 94720.
Sticher
L
L
van Huystee
R
R
Wagner
D
D
Jones
R L
RL
D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002942:Circular Dichroism; D004795:Enzyme Stability; D001467:Hordeum / Q000201:enzymology; D015759:Ionomycin / Q000494:pharmacology; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D010944:Plants / Q000201:enzymology*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D000516:alpha-Amylases / Q000302:isolation & purification / Q000378:metabolism*
D007527:Isoenzymes; D000001:Calcimycin; D015759:Ionomycin; D000516:alpha-Amylases; D002118:Calcium