Purification of carbonic anhydrase isozyme VI (CA-VI) from swine saliva.
Salivary or secreted carbonic anhydrase (CA), which constitutes a new class of CA, designated CA-VI, was isolated. Swine CA-VI purified from swine saliva by inhibitor-affinity chromatography and ion exchange chromatography had a specific activity of 5,468 units/mg. The molecular weight was 250,000, as determined by gel filtration under non-denaturing conditions, and the subunit molecular weight was found to be 37,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that swine CA-VI consists of 7 subunits. The treatment of the enzyme with endo-N-acetylglucosaminidase F reduced its subunit molecular weight from 37,000 to 35,000 and 32,000. We raised a rabbit antibody against purfied swine CA-VI. Double immunodiffusion showed that anti-swine CA-VI serum reacted with swine CA-VI and swine saliva, but not with hemolysate (containing CA-I and CA-Il) or muscle extracts (containing CA-III). The concentration of CA-VI in swine saliva, measured using single radial immunodiffusion, was 0.027 +/- 0.017 mg/mg total protein.
Nishita
T
T
Laboratories of Veterinary Physiology 1, Azabu University, Sagamihara, Kanagawa, Japan.
Sakomoto
M
M
Ikeda
T
T
Amasaki
H
H
Shino
M
M
D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000662:veterinary; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D005779:Immunodiffusion / Q000662:veterinary; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification; D008297:Male; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry; D008970:Molecular Weight; D012463:Saliva / Q000201:enzymology*; D012471:Salivary Proteins and Peptides / Q000737:chemistry / Q000302:isolation & purification*; D013552:Swine / Q000378:metabolism*
D007527:Isoenzymes; D012471:Salivary Proteins and Peptides; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D002256:Carbonic Anhydrases