ABSTRACT
Carbonic anhydrase (CA) from erythrocytes of the pink salmon, Onchorhyncus gorbushka, was purified using chloroform-ethanol extraction and Sephadex G-75 gel filtration. A single, high specific-activity CA isozyme having a molecular weight of 29,000 was found. The enzyme sedimented as a single boundary at a sedimentation velocity of 2.9S. Amino acid analysis revealed a composition similar to other submammalian CAs with the exception that the cysteine content was low (1 mol cysteine/mol enzyme). Like other submammalian CAs, the presence of a sulfhydryl reducing agent was required to maintain full activity and to prevent structural changes in the enzyme.
Subject(s)
Carbonic Anhydrases/blood , Erythrocytes/enzymology , Amino Acids/analysis , Animals , Carbonic Anhydrases/isolation & purification , Dithionitrobenzoic Acid/pharmacology , Kinetics , Molecular Weight , Salmon , Species SpecificityABSTRACT
1. A single, high specific activity carbonic anhydrase (CA) isozyme was present in erythrocytes of the teleostean species Salmo gairdneri (rainbow trout). 2. Purification of trout CA to homogeneity was accomplished using chloroform ethanol extraction, Sephadex G-75 gel filtration, and DEAE Bio-Gel anion exchange chromatography. 3. Trout CA was a zinc metalloenzyme of mol. wt 28,300 and pI9.3. 4. Amino acid analysis indicated the presence of 6 half-cystine residues per enzyme molecule, and the presence of a sulfhydryl reducing agent was required to maintain full activity in vitro. 5. Sulfhydryl modification with both N-ethylmaleimide and acrylonitrile indicated the presence of 3 reactive sulfhydryl groups per CA molecule. Modification of those groups had no direct effect on enzyme activity, but modified CA was no longer subject to inactivation by oxidizing conditions.
Subject(s)
Carbonic Anhydrases/blood , Erythrocytes/enzymology , Isoenzymes/blood , Salmonidae/blood , Trout/blood , Acetazolamide/pharmacology , Amino Acids/analysis , Animals , Carbonic Anhydrase Inhibitors/pharmacology , Carbonic Anhydrases/isolation & purification , Chemical Phenomena , Chemistry , Chemistry, Physical , Spectrum Analysis , Structure-Activity Relationship , Zinc/analysisABSTRACT
1. Solutions of purified carbonic anhydrase from chicken and salmon erythrocytes were incubated in buffer for 1 hr at 5 degrees or 22 degrees C, at pH 7.3, 7.6, 7.9 or 8.2. 2. At 22 degrees C the enzymes lost up to 25% of their ability to catalyze the CO2 hydration reaction when compared to control solutions maintained in the cold. 3. Loss of enzyme activity (approximately 50%) also occurred as pH was increased from 7.3 to 8.2. 4. The presence of lecithin vesicles or microsomes completely protected the enzymes from denaturation caused by pH changes and afforded partial protection from changes due to increased temperature.
Subject(s)
Carbonic Anhydrases/analysis , Chickens/metabolism , Membrane Lipids/physiology , Salmon/metabolism , Animals , Carbon Dioxide/metabolism , Female , Hydrogen-Ion Concentration , Membranes, Artificial , Microsomes/enzymology , TemperatureABSTRACT
1. High activity (CA C) and low activity (CA B) carbonic anhydrase isoenzymes have been purified from turtle erythrocytes. 2. The two isoenzymes differed in CO2 hydration specific activity by 36-fold. 3. The low activity isoenzyme contained one half-cystine residue, whereas the high activity isoenzyme contained four half-cystines and required a reducing environment to maintain activity. Both isoenzymes contained zinc. 4. Molecular weights of 28,500 and 30,400 daltons were established for the low and high activity isoenzymes respectively. 5. Both isoenzymes were inhibited by acetazolamide, but only the high activity isoenzyme was inhibited by parachloromercuribenzoate. 6. The low activity isoenzyme was present in the erythrocytes at about 8-10 times the concentration of the high activity isoenzyme. 7. The high activity isoenzyme cross-reacted with antibodies prepared against pure chicken carbonic anhydrase C.
