The purification of alkaline phosphatases of animal tissues

Abstract

Cyclization of the chloroacetyl derivative was effected by dissolving it (5-36 g.) in hot ethanol (50 ml.) and diluting with water (140 ml.). When the solution became clear again, NaOH (115 g.) in water (10 ml.) was added, and a yellow colour developed owing to partial hydrolysis. The solution was refluxed for 30 min. and acidified with HCI while still hot. On cooling, colourless, chlorine-free, prismatic needles separated and were collected (4.3 g., 72% overall yield, m.p. 240-242°). Crystallization from ethanol gave synthetic viridicatin, m.p. 264-265', not depressed on admixture with natural viridicatin from P. cydopium.

Studies on the fractionation of mucosal homogenates from the small intestine

“…had come to the same conclusion in their investigation of rabbit small-intestinal mucosa. With the exception of the work of Morton (1954), who discarded the first low-speed sediment in his fractionations, no explanation can be given that might reconcile these opposing views.…”

“…The reports on the intracellular distribution of alkaline phosphatase are somewhat more contradictory. A predominantly microsomal localization of this enzyme was reported for the intestinal mucosa of the rat (Allard, de Lamirande & Cantero, 1957;Triantaphyllopoulos & Tuba, 1959;Robinson, 1963;Ailhaud et al 1963), the calf (Morton, 1954) and the rabbit and guinea pig (Hers, Berthet, Berthet & de Duve, 1951). In contrast, Holt & Miller (1962) had described alkaline phosphatase as a typical brush-border enzyme in the intestinal mucosa ofthe hamster.…”

“…The fractionation of homogenates of the mucosa of the small intestine has been described for preparations from the rat (Schmidt, Bessman & Thannhauser, 1957;Busch, Davis & Anderson, 1958;Triantaphyllopoulos & Tuba, 1959;Epstein & Shapiro, 1959;Senior & Isselbacher, 1962;Ailhaud, Samuel & Desnuelle, 1963;Robinson, 1963), the hamster (Brown & Johnston, 1964), the guinea pig (Glover & Green, 1957;Sherratt & Hubscher, 1963), the rabbit (Doell & Kretschmer, 1962;Carnie & Porteous, 1962b;, the cat (Hubscher, Clark, Webb & Sherratt, 1963), the dog (Baker, 1958), the pig (Buell & Reiser, 1959; Borgstrom & Dahlqvist, 1958), the calf (Morton, 1954) and man (Doell & Kretschmer, 1962). The suspending media described in these procedures vary in composition, and include salt solutions (Schmidt et al 1957; Buell & Reiser, 1959), 0-25M-or 03M-sucrose (Busch et al 1958;Baker, 1958;Hubscher et al 1963) and buffered mannitol solutions (Senior & Isselbacher, 1962).…”

Rat intestinal alkaline phosphatase. A microheterogeneous series of glycoproteins

“…The purification procedure consisted essentially of the following steps:-butanol extraction [6] , fractional precipitation with ethanol, chromatography on DE 11 cellulose (Whatman), gel filtration on Sephadex G-200 (Pharmacia) and finally chromatography on DE 32 cellulose (Whatman).…”

Some factors controlling alkaline phosphatase isoenzymes in HeLa cells

“…This induction is believed to involve a change in conformation of the enzyme rather than an increase in the amount of enzyme (Griffen & Cox, 1966 (1958) and used at 0.5mg/ml. The cells were harvested by trypsinization and homogenates prepared by Morton's (1954) (Lowry, Rosebrough, Farr & Randall, 1951).The isoenzymes were separated by polyacrylamide-gel electrophoresis (Spencer, 1967 (Dulbecco & Freeman, 1959) or by Waymouth's (1959) (0.5mg/ml). After 6 days in this medium the electrophoretogram showed the 'calf-serum' pattern but with a very weak slow band.…”