Elizabeth W. Bingham

Removal of phosphate groups from casein with potato acid phosphatase.

Potato acid phosphatase (EC 3.1.3.2) was used to remove the eight phosphate groups from alphas1-casein. Unlike most acid phosphatases, which are active at pH 6.0 or below, potato acid phosphatase can catalyze the dephosphorylation of alphas1-casein at pH 7.0. Although phosphate inhibition is considerable (K1=0.42 mM phosphate), the phosphate ions produced by the dephosphorylation of casein can be removed by dialysis, allowing the reaction to go to completion. The dephosphorylated alphas1-casein is homogeneous on gel electrophoresis with a slower mobility than native alphas1-casein and has an amino acid composition which is identical to native alphas1-casein. Thus the removal of phosphate groups from casein does not alter its primary structure. Potato acid phosphatase also removed the phosphate groups from other phosphoproteins, such as beta-casein, riboflavin binding protein, pepsinogen, ovalbumin, and phosvitin.

Purification and properties of acid phosphatase in bovine milk

Abstract The acid phosphatase of bovine milk has been purified over 40,000-fold by extensive use of Amberlite IRC-50. The enzyme is a basic protein with an optimum pH of 4.75. Ascorbic acid triples the activity, while other reducing agents have only a slight activating effect. The acid phosphatase acts on aromatic phosphates, casein, and pyrophosphates (both organic and inorganic). It did not act on AMP, serine phosphate, and glycerol phosphate.

Ribonuclease in bovine milk

Abstract The ribonuclease of milk has not been investigated, although there has been in recent years a rapid expansion of our knowledge of ribonuclease in body fluids such as cerebral spinal fluid ( Houck, 1958 ), blood ( Levy and Rottino, 1960 ; Rabinovitch and Dohi, 1957 ; Zittle and Reading, 1945 ), urine ( Levy and Rottino, 1960 ), and the extracellular fluid of skin ( Tabachnick and Freed, 1961 ). Zittle and DellaMonica (1952) noted that certain purified fractions of bovine milk showed phosphodiesterase activity when ribonucleic acid was used as the substrate. Bailie and Morton (1958) showed that the nucleic acid content of mammary gland microsomes diminished when the microsomes were incubated in milk serum for 12 hours, and suggested that the phosphodiesterase of Zittle and DellaMonica (1952) might have caused the decrease in nucleic acid. They suggested that milk microsomes (with a low nucleic acid content) might be derived from mammary gland microsomes, which contained a much higher nucleic acid content. In this report evidence is presented for the presence of relatively high concentrations of ribonuclease in cows milk. Some properties of this enzyme, as well as its partial purification, are described.

Ribonuclease B of bovine milk

Abstract Ribonuclease B has been isolated from 757 liters of milk; pilot plant facilities were used to provide a crude fraction. Final purification was achieved by gel filtration on Sephadex G-75 and chromatography on IRC-50. Milk ribonuclease A was also isolated by this procedure. Milk ribonuclease B has an amino acid composition which is identical to pancreatic ribonuclease A, milk ribonuclease A, and pancreatic ribonuclease B. It is a glycoprotein containing 4.2% hexosamine (3.0% glucosamine plus 1.2% galactosamine) and 5.17% mannose. It differs from pancreatic ribonuclease B, which contains 2.16% glucosamine, no galactosamine, and 5.7% mannose.

Alkaline phosphatase in the lactating bovine mammary gland and the milk fat globule membrane. Release by phosphatidylinositol-specific phospholipase C

1. Alkaline phosphatase is covalently bound to bovine mammary microsomal membranes and milk fat globule membranes through linkage to phosphatidylinositol as demonstrated by the release of alkaline phosphatase following treatment with phosphatidylinositol-specific phospholipase C. 2. The release of alkaline phosphatase from the pellet to the supernatant was demonstrated by enzyme assays and electrophoresis. 3. Electrophoresis of the solubilized enzymes showed that the alkaline phosphatase of the microsomal membranes contained several isozymes, while only one band with alkaline phosphatase activity was seen in the fat globule membrane. 4. Levamisole and homoarginine were potent inhibitors of the alkaline phosphatase activities in both membrane preparations and in bovine liver alkaline phosphatase, but not in calf intestinal alkaline phosphatase.

Phosphorylase kinase from rabbit skeletal muscle: Phosphorylation of κ-casein

Abstract Phosphorylase kinase (EC 2.7.1.38) from rabbit skeletal muscle catalyzed the phosphorylation of κ-casein (Mr = 19,000) to a stoichiometry of approximately one mole of phosphate per mole of κ-casein. The reaction rate was modified by several factors known to influence phosphorylase kinase activity: (1) stimulation by Ca2+, (2) further stimulation by added calmodulin in the presence of Ca2+, (3) higher activity at pH 8.2 than at pH 6.8, and (4) activation, measured at pH 6.8, following partial proteolysis of the kinase by trypsin. The maximal rate of phosphorylation of κ-casein by nonactivated phosphorylase kinase in the presence of Ca2+ at pH 8.2 was 16 nmol/min/mg, and the κ-casein concentration for half-maximal activity was 80 μ m . The phosphorylation of other components of whole casein, αs1-casein and β-casein, by phosphorylase kinase was detectable but much slower than the reaction with κ-casein. When whole casein was used as a substrate, κ-casein was identified by electrophoresis as a major phosphorylated species. The amino acid residues in κ-casein modified by phosphorylase kinase were shown to be serines. The present work extends the known substrates of phosphorylase kinase to include a well-characterized protein that may prove an interesting model substrate. Furthermore, this report emphasizes that whole casein, because of its heterogeneity, is a poor substrate to use in characterizing the substrate specificities of protein kinases, since different “casein kinases” may be specific for different components of whole casein.

Phosphorylation of casein by cyclic AMP-dependent protein kinase

Abstract The catalytic subunit of rabbit muscle cyclic AMP-dependent protein kinase (EC 2.7.1.37; ATP:protein transferase) has been tested on a variety of caseins. The B variant of β-casein was phosphorylated at a much greater rate than other β-caseins, αs1-caseins, and κ-caseins. Whole casein homozygous for β-casein B was phosphorylated at 2.5 times the rate of commercial whole casein. Gel electrophoresis experiments indicate that β-casein is the predominant component phosphorylated in commerical casein. It is therefore suggested that phosphorylation of whole casein depends on its content of the specific genetic variant, β-casein B.

Reactivity of the alkaline phosphatases of bovine milk and intestinal mucosa with the substrates phenyl phosphate and o-carboxyphenyl phosphate.

Abstract Bovine intestinal mucosa alkaline phosphatase reacts equally with the substrates phenyl phosphate and o -carboxyphenyl phosphate. Milk alkaline phosphatase, on the other hand, at a concentration of the substrates of about 8 × 10 −5 moles/l., is 1 3 to 1 4 reactive with the o -carboxyphenyl phosphate as with phenyl phosphate. Analyses of these reactions with a range of substrate concentrations showed that the difference in reactivity is due to a difference in enzyme-substrate dissociation constants, K m . The p K m values for the mucosa phosphatase with both substrates at pH 9.7 is 3.7; with the milk phosphatase and phenyl phosphate the p K m value is 4.6, whereas with o -carboxyphenyl phosphate the value is 2.8. The K m values were also determined for a range of pH values. The difference noted above persisted throughout. K m values obtained with the milk phosphatase in a lipide-containing complex were identical with values obtained with the phosphatase after dissociating with n -butyl alcohol.

Influence of magnesium ion on the ultraviolet absorption of aqueous solutions of salicylic acid and related compounds

Abstract The influence of magnesium ion on the ultraviolet absorption of salicylic acid and related compounds has been studied. Both salicylic acid and methyl salicylate are affected by the presence of magnesium ions. There is an increase in the absorption maximum, and the maximum shifts to longer wavelengths. This effect is marked only at higher pH values.