The Fat-Globule Membrane of Milk: Alkaline Phosphatase and Xanthine Oxidase in Skimmilk and Cream

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Recent studies of Morton (11,1,3) show that the fat-globule membrane of milk has many properties in common with the enzyme-containing, lipoprotein complexes of aninml tissues termed microsomes.The complex nature of the fat-globule membrane suggests that isolation methods involving pasteurization(3), the use of low pH (6), and lipide-solvents (3, 6) should be avoided to isolate this material in its native state.Further, by avoiding pasteurization, enzyme assays (xanthiue oxidase and alkaline phosphatase in the present studies) can be used to follow the fractionation.In the present report the lipoprotein fraction from cream is compared with a similar fraction from skimnlilk in behavior in the ultracentrifuge and in filtrability, chemical composition, and enzyme content. METHODSAlkaline phosphatasc.Phenol phosphate is used as substrate and the assay is performed at pit 9.6 in ethanolamine buffer at 37 ° C. (18).Enzyme activities are expressed in terms of optical absorbance in an 18 mm.diameter calibrated test tube at 660m~.An absorbanee of 0.]05 (10.5 units) is equivalent to the release of 1.0 × 10 7 moles of phenol. Xanthine oxidasc.The method is based on the ability of xanthine oxidase to catalyze the reduction of the colorless triphenyltetrazolium chloride to a red substance when xanthine or other suitable substrate is present.Details of the method are described elsewhere (17).The formation of 3 × 10 -z moles of reduced triphenyltetrazolium under the conditions of the test is defined as 10 units of oxidase. Determination of lipides by extraction with orgasmic solve~ts.Five hundred to 800 mg. of freeze-dried solids were extracted twice with 40 ml.acetone and finally with 40 ml.ethyl ether.When it was desired to dissociate the lipoprotein (10), the acetone extraction was followed by 40 ml.~-butanol and finally with ethyl ether. Determination of nitroge~ a~d protein.The nitrogen content was determined by the Nessler method.Comparative protein estimations were made routinely with the biuret reagent. Determination of phosphorous.Samples were digested with sulfuric acid and hydrogen peroxide.Phosphorus was estimated with the ammonimn molybdate reagent after reduction with ferrous sulfate.