Selective extraction of alkaline phosphatase and 5'-nucleotidase from milk fat globule membranes by a single phase n-butanol procedure.
A single phase extraction procedure employing 8% (v/v) n-butanol at room temperature extracted over 90% of alkaline phosphatase activity and over 60% of 5'-nucleotidase activity from bovine milk fat globule membranes (MFGM). For 5'-nucleotidase, higher n-butanol concentrations lead to loss of activity, while lower concentrations were ineffective in extracting the enzyme. When extractions were performed at 0 degrees C, similar yields were obtained for alkaline phosphatase extraction with 8% (v/v) n-butanol, but 5'-nucleotidase extraction required 10% (v/v) n-butanol for similar yields. However, 5'-nucleotidase was less susceptible to denaturation during extraction at 0 degrees C. The Km values and substrate specificities for both alkaline phosphatase and 5'-nucleotidase were unchanged by extraction with 8% (v/v) n-butanol. The 8% (v/v) n-butanol extraction procedure provides a 3-fold purification step, and an enzyme preparation suitable for further purification.
Ahn
Y S
YS
Department of Chemistry, Louisiana Tech University, Ruston 71272.
Snow
L D
LD
D020001:1-Butanol; D015720:5'-Nucleotidase / Q000187:drug effects / Q000302:isolation & purification*; D000469:Alkaline Phosphatase / Q000187:drug effects / Q000302:isolation & purification*; D000818:Animals; D000440:Butanols / Q000494:pharmacology; D002417:Cattle; D003080:Cold Temperature; D004305:Dose-Response Relationship, Drug; D006358:Hot Temperature; D008566:Membranes / Q000187:drug effects / Q000201:enzymology; D008892:Milk / Q000201:enzymology*; D011489:Protein Denaturation; D013379:Substrate Specificity
D000440:Butanols; D020001:1-Butanol; D000469:Alkaline Phosphatase; D015720:5'-Nucleotidase