The banding pattern of rat liver carboxylesterases (EC 3.1.1.1) was demonstrated following polyacrylamide gel electrophoresis and isoelectric focusing using standardized conditions. Phenotypic variations, occurring in commonly used inbred rat strains, were compared. Separate isozymes were identified using genetic nomenclature. Individual bands were labelled; their electrophoretic parameters were estimated. Three hitherto genetically undefined zones were dessribed and preliminarily classified as carboxylesterase isozymes. A scheme was provided to enable identification of liver esterases in rat strains not investigated in the present study.
Esterase 6A was isolated from mouse lung and purified 440-fold by ion-exchange chromatography, inverse ammonium sulphate gradient solubilization, gel filtration and isoelectric focusing. The resultant product was apparently homogenous by the criteria of polyacrylamide gel electrophoresis and immunodiffusion, and consisted of the electrophoretic form 6A3. A single species of subunit was present on sodium dodecyl sulphate gel electrophoresis. The molecular weight of the native protein was found to be about 178000 with a subunit molecular weight of about 60000. The equivalent weight obtained by active-site titration with diethyl-p-nitrophenyl phosphate was approximately 178 000 g/mol, indicating a functional asymmetry in the trimer. The enzyme was shown to have a high affinity for 4-nitrophenyl hexanoate (Michaelis constant K , = 4.4 pmol/l) with a relatively low catalytic efficiency (catalytic constant k,,, = 12 s-'). Esterase 6A was immunologically related to esterase 1 and esterase 9, with which it is genetically closely linked. Further properties of the three esterases were compared.
Isozymes of chloroplast glyceraldehyde‐3‐phosphate dehydrogenase (GPD, EC 1.2.1.13) from Chenopodium rubrum were separated using inverse discontinuous ammonium sulphate gradient solubilization. Leaves were extracted at the 9th h of light and the 9th h of darkness of a 12 h light/12 h dark cycle. The ratio of “NADP‐GPD I” to “NADP‐GPD II” varied with the light/dark cycle. However, the “light” isozyme pattern could be obtained from “dark” plants by including NADP + or NAD + kinase in the extraction buffer. Similarly, the “dark” isozyme pattern was produced in “light” plants extracted in the presence of NAD+. Pyridine nucleotides had no effect on the separated, purified isozymes. It is concluded that differential binding of the isozymes at the moment of extraction to pelletable material in the crude extract determines the isozyme pattern, and that this binding is regulated by the pyridine nucleotide ratio.
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