Conjugases preparation

The kinetics of hog kidney conjugase with crystallized PGA conjugate as substrate have been studied by Bird et al. (72). These workers employed an enzyme preparation, obtained as follows. Kidney homogenate, prepared in a Waring Blender, is suspended in 3 times its weight of water, centrifuged, and then filtered. The activity of this filtrate is stable in the cold. This conjugase preparation is not activated by calcium ions, and its pH optimum is close to 4.5. 

Conjugase responds rapidly to zinc depletion and repletion, and it has been suggested that the absorption of a test dose of folate polyglutamates may provide a sensitive index of zinc nutritional status (Canton and Cremin, 1990). The absorption of folate monoglutamates (from pharmaceutical preparations or foods) is not affected. 

The hypothesis that contraceptive steroids might interfere with folate deconjugation in the small intestine was examined by Stephens et al. (S19). Homogenized human jejunal mucosa obtained by peroral biopsy and lysosomal preparations from guinea pig jejunum were utilized. Estradiol, estrone, and progesterone were found not to inhibit folate conjugase in these systems, or to inhibit transport of folate conjugase across lysosomal membranes. 

The same technique applies to the preparation of vitamin B from horse liver, which is even richer than hog liver in this vitamin (459). The preparation of vitamin B, from yeast is carried out by the same technique, after a treatment of the yeast extract with conjugase. The crystallized product obtained from yeast is identical with that prepared from liver. Stokstad et al. (557) isolated the liver L. casei factor from the precipitate obtained by treating the aqueous liver extract with 85% ethanol. The active substance is separated by successive adsorption on norit A and Superfiltrol, the last eluate being precipitated by baryta and ethanol. The L. casei factor is esterified, and the esterified product is extracted by butanol. After removal of butanol, the substance is dissolved in methanol and chromatographed on a column of Superfiltrol. It is then eluted with 70 % aqueous acetone. The active esterified product thus eluted is pure, and on hydrolysis yields the free acid which crystallizes. The latter is identical to the vitamin Bo of Pfiffner et al. (459). 

A suspension of 5 grams of brewers yeast in 100 ml. of distilled water was autoclaved at 110° for 30 minutes. After centrifugation, the supernatant liquid was the source of the CF conjugate. On incubation of the liquid for 12 hours with a preparation of conjugase from hog kidney, the Citrovorum factor activity increased threefold. 

Mims and Laskowski (416) have described a technique for preparing a highly purified chicken pancreas conjugase. 

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