Enhancement of carbonic anhydrase activity by erythrocyte membranes.
The human erythrocyte membrane is an efficient enhancer of both high (CA II) and low (CA I) activity isozymes of red blood cell carbonic anhydrase. The presence of membrane increased CO2 hydration catalyzed by bovine CA II 1.6-fold, human CA II 3.5-fold, and human CA I 1.6-fold. With the high activity CA isozymes, maximal stimulation was observed in the presence of 1-3 micrograms membrane protein/ml. The Vmax for bovine CA II (4 nM) rose from 0.302 to 0.839 mM/s, while that for human CA II (6 nM) increased from 0.113 to 0.414 mM/s in the absence and presence of membrane, respectively. The apparent Km for CO2 increased from 13.2 to 51.2 mM for bovine CA II, and from 6.5 to 38.5 mM for human CA II. Mixtures of membrane plus enzyme, upon centrifugation through linear sucrose density gradients, displayed enhanced Ca activity only in membrane-containing gradient fractions, verifying the stimulatory ability of membranes on enzyme activity and indicating tight and stable complex formation. Membrane enhancement of CA activity appears to be a general phenomenon in that mouse hepatocyte membranes also stimulated CA activity, although less efficiently than erythrocyte membranes. Of the many soluble putative effectors assayed, only imidazole enhanced CA II activity to an extent comparable with erythrocyte membranes; imidazole did not, however, stimulate the activity of human CA I. The data are consistent with a model of CA II activation by membrane association that may effect a distortion of the enzyme conformation in such a way as to facilitate intra- and/or intermolecular proton transfer between membrane-bound and enzyme-bound proton shuttling residues (perhaps the imidazole moiety of histidine) and the Zn-bound hydroxide at the catalytic site of the enzyme.
Parkes
J L
JL
Department of Environmental Medicine, New York University Medical Center, New York 10016.
Coleman
P S
PS
D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D002256:Carbonic Anhydrases / Q000097:blood*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000502:physiology*; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007527:Isoenzymes / Q000097:blood*; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008297:Male; D008563:Membrane Lipids / Q000494:pharmacology; D051379:Mice; D008809:Mice, Inbred C3H; D010455:Peptides / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology; D011485:Protein Binding
D000596:Amino Acids; D007527:Isoenzymes; D008563:Membrane Lipids; D010455:Peptides; D010743:Phospholipids; D002256:Carbonic Anhydrases