The properties of purified low molecular weight phosphoprotein phosphatase from rabbit heart.

A simplified procedure for the purification of low molecular weight phosphoprotein phosphatase acting on muscle phosphorylase a has been described from rabbit heart. The enzyme was purified to homogeneity by acid precipitation, ethanol treatment, and chromatography on Sephadex G-75 and Sepharose-histone. The purified enzyme showed a single band when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the molecular weight calculated by this method was 34 000. The S20, W value and Stokes radius for the enzyme was 3.35 and 24.0 A(1 A = 0.1 nm), respectively. Using these two values, a molecular weight of 35 000 was calculated. Purified enzyme showed a wide substrate specificity and catalyzed the dephosphorylation of phosphorylase a, glycogen synthase D, phosphorylated histone, and phosphorylated casein. Kinetic studies revealed the lowest Km with glycogen synthase D and maximum Vmax for the reaction with phosphorylase a.     

Some properties of purified 3-hydroxy-3-methylglutaryl coenzyme A reductase phosphatases from rat liver

Incubation of four purified rat liver 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase phosphatases (G. Gil, M. Sitges, and F. G. Hegardt, (1981) Biochim. Biophys. Acta663, 211–221) with HMG-CoA, CoA, NADPH, or citrate caused a concentration-dependent inactivation of the enzyme activities. HMG-CoA and CoA showed similar patterns of inactivation and at 0.5 mm of both compounds, the four reductase phosphatases were fully inhibited. Half-maximal inactivation was comprised between 0.02 and 0.1 mm of HMG-CoA and CoA. NADPH at concentration ranging between 5 and 10 mm produced complete inactivation of reductase phosphatases. Citrate at 5 mm produced full inactivation, and half-maximal inhibition ranged from 0.1 to 0.4 mm for the different phosphatases. The behavior of fluoride varied with respect to the four phosphatases: Low molecular forms were inactivated in a similar manner as described for other protein phosphatases. However, high molecular forms were slightly inactivated, and phosphatase IIa at 100 mm showed a level of activity similar to the control. The effect of KCl on the four reductase phosphatases could explain this behavior since at high concentrations, KCl (and NaCl) produced activation in both high and low molecular forms, this effect being more enhanced in high M_r reductase phosphatases. The insensitivity to fluoride of high M_r reductase phosphatases could explain the discrepancies in percentage of the active form of HMG-CoA reductase described previously in literature.     

Purification and properties of a heat-stable protein inhibitor of phosphoprotein phosphatase from rabbit liver.

A heat-stable protein inhibitor of phosphoprotein phosphatase has been purified to homogeneity from rabbit liver extract by heating to 95 degrees followed by ion exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The purified inhibitor showed a single band when examined by gel electrophoresis S20, w and Stokes radius values were 1.45 and 25.5, respectively. Using these two values, the molecular weight and frictional ratio was calculated to be 15,500 and 3.40, respectively. The molecular weight determined by sodium dodecyl sulfate-gel electrophoresis was found to be 14,200. The inhibition of phosphoprotein phosphatase was linear up to 40% inhibition with respect to inhibitor was constant with time of incubation for at least 30 min. The optimum pH for the inhibition was between 6.8 and 7.6. A kinetic analysis of the effect of the inhibitor on the dephosphorylation of [32P]phosphorylase a by rabbit liver phosphoprotein phosphatase indicated a noncompetitive inhibition with respect to phosphorylase a. Purified liver inhibitor inhibited the phosphoprotein phosphatase activity in all rat tissues examined. Utilizing purified rabbit liver phosphoprotein phosphatase, the presence of inhibitor activity was also demonstrated in all rat tissues tested.     

Some kinetic properties of gamma-glutamyltransferase from rabbit liver

gamma-Glutamyltransferase ((5-glutamyl)-peptide: amino-acid 5-glutamyltransferase, EC 2.3.2.2) of rabbit liver (detergent form) was purified 1100-fold in order to study its kinetic properties. Kinetic studies were conducted from pH 6.0 to 12.0 in the absence and presence of the acceptor substrate glycylglycine using gamma-glutamyl-3-carboxy-4-nitroanilide as the donor. The existence of more than one binding site for both donor and acceptor is postulated on kinetic evidence such as donor substrate activation, donor substrate inhibition and acceptor substrate activation. Homotropic interaction is also observed, in the form of negative cooperativity, in donor substrate binding, in the absence of acceptor at pH less than 9.0 and positive cooperativity (n = 2), in the absence or presence of acceptor at pH greater than 9.0. Hydrolase reaction reaches a maximum of activity at pH 10 (pK 8.6). Transferase activity under conditions of maximal velocity is maximal at pH 9.0 (pK 7.1). The ratio of transferase activity/hydrolase activity is maximal at pH 7.0-7.5. At low donor substrate concentrations, maximal activity is attained at pH 7.5.     

Some properties of phosphatidylcholine exchange protein purified from beef liver

A phospholipid exchange protein has been purified 2680-fold from beef liver. The assay of the exchange activity of the protein was based on the transfer of [¹⁴C]phosphatidylcholine from microsomes labeled with [¹⁴C]phosphatidylcholine to liposomes. The homogeneity of the protein has been established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoelectrophoresis and isoelectric focusing. The protein has a molecular weight of approximately 22000 and an isoelectric point of 5.8. The amino acid composition has been determined. The protein contains one disulfide bridge and has glutamic acid as the N-terminal amino acid. Phospholipid, tentatively identified as phosphatidylcholine, was found to be present in the protein preparation. The protein stimulated specifically the exchange of phosphatidylcholine between mitochondria and microsomes from rat liver.     

Purification, properties, and substrate specificities of phosphoprotein phosphatase(s) from rabbit liver.

The phosphoprotein phosphatase(s) acting on muscle phosphorylase a was purified from rabbit liver by acid precipitation, high speed centrifugation, chromatography on DEAE-Sephadex A-50, Sephadex G-75, and Sepharose-histone. Enzyme activity was recovered in the final step as two distinct peaks tentatively referred to as phosphoprotein phosphatases I and II. Each phosphatase showed a single broad band when examined by sodium dodecyl sulfate gel electrophoresis; the molecular weights derived by this method were approximately 30,500 for phosphoprotein phosphatase I and 34,000 for phosphoprotein phosphatase II. The s20, w value for each enzyme was 3.40. Using this value and values for the Stokes radii, the molecular weight for each enzyme was calculated to be 34,500. Both phosphatases, in addition to catalyzing the conversion of phosphorylase a to b, also catalyzed the dephosphorylation of glycogen synthase D, activated phosphorylase kinase, phosphorylated histone, phosphorylated casein, and the phosphorylated inhibitory component of troponin (TN-I). The relative activities of the phosphatases with respect to phosphorylase a, glycogen synthase D, histone, and casein remained essentially constant throughout the purification. The activities of both phosphatases with different substrates decreased in parallel when they were denatured by incubation at 55 degrees and 65 degrees. The Km values of phosphoprotein phosphatase I for phosphorylase a, histone, and casein were lower than the values obtained for phosphoprotein phosphatase II. With glycogen synthase D as substrate, each enzyme gave essentially the same Km value. Utilizing either enzyme, it was found that activity toward a given substrate was inhibited competitively by each of the alternative substrates. The results suggest that phosphoprotein phosphatases I and II are each active toward all of the substrates tested.     

Purification and properties of a phosphoprotein phosphatase from rat liver.

A phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) has been partially purified from rat liver homogenates by (NH4)2SO4 and ethanol precipitations followed by DEAE-cellulose and Sepharose 6B chromatography. The phosphoprotein phosphatase is capable of cleaving [32P]phosphate from radiolabelled phosphopyruvate kinase (type L) (EC 2.7.1.40), phosphohistones, and phosphoprotamine. However, it did not detectably dephosphorylate ATP, ADP, DL-phosphorylserine or beta-glycerophosphate. Dephosphorylation of [32P]phosphopyruvate kinase was stimulated by divalent cations and inhibited by ATP, ADP, Fru-1,6-P2, and orthophosphate. Divalene cations could reverse inhibition induced by ADP or ATP. At least one function of the phosphoprotein phosphatase may be to remove phosphate groups from the phosphorylated form of pyruvate kinase in the liver.     

Characterization of nuclear phosphoprotein phosphatases from goat testis

Two nuclear phosphoprotein phosphatases (PPases I and II) that cause dephosphorylation of [32P]histone, have been partially purified from goat testis. The enzymic activity is associated with nucleoplasm and chromatin. PPase I is markedly stimulated (approx. 200-600%) by Mg2+ or Mn2+ (1 mM) whereas Ca2+ (1 mM) causes slight stimulation (approx. 35%) of the enzyme. On the contrary, PPase II is only slightly activated (20-40%) by these metal ions (5 mM). Both the phosphoprotein phosphatase isoenzymes are maximally active at pH 6-7. PPases I and II are strongly inhibited (approx. 60-100%) by ZnCl2 (1 mM), P1 (5 mM) and thiol reagents. NaF (5 mM) inhibits (approx. 40%) specifically the activity of PPase I rather than PPase II. PPases are strongly inhibited by relatively high concentration of NaCl (0.4 M), isoenzyme II being more sensitive (approx. 80%) than isoenzyme I (approx. 50%). In addition to histones, both the isoenzymes can as well cause dephosphorylation of protamine, casein, and testicular nuclear proteins. Enzymic characteristics of the testicular nuclear PPases are clearly different from those of the cytosolic enzyme previously characterized.     

Sequence homology between purple acid phosphatases and phosphoprotein phosphatases. Are phosphoprotein phosphatases metalloproteins containing oxide-bridged dinuclear metal centers?

The amino acid sequences of mammalian purple acid phosphatases and phosphoprotein phosphatases are shown to possess regions of significant homology. The conserved residues contain a high percentage of possible metal-binding residues. The phosphoprotein phosphatases 1, 2A and 2B are proposed to be iron-zinc metalloenzymes with active sites isostructural (or nearly so) with those of the purple phosphatases.     

Purification and characterization of two phosphorylase phosphatases from rabbit liver

Two phosphorylase phosphatase activities (I and III) have been purified from rabbit liver, with respective molecular weights of 117,000 and 230,000. Phosphatase III contained three different subunits of molecular weights 35,000, 67,000 and 80,000. Phosphatase I although majoritary in the preparation, was not homogeneous. Both phosphatases were dissociated by 2-mercaptoethanol treatment, releasing a catalytic subunit with a molecular weight of about 35,000. Phosphatases I and III activities responded very differently to incubation with trypsin and to ethanol precipitation. Phosphatase III was much more sensitive to inactivation by several ions and ATP than phosphatase I. On the basis of the obtained data, phosphatase I can be classified as a type-1 phosphatase and phosphatase III as a type-1 phosphatase.     

Some properties of partially purified phytase from Aspergillus niger.

Phytase was purified from Aspergillus niger culture fluid by molecular sieve filtration on Sephadex G-200, followed by thermal inactivation of acid phosphatase and CM-cellulose chromatography. The 12-fold purified enzyme had two pH optima at 2.7 and 5.5 and was characterized by high thermal stability in alkaline environment and broad substrate specificity. The Michaelis constant of phytase relative to myo-inositol hexaphosphate sodium salt is 4.8 X 10(-4) M and activation energy 9,217 cal/mole. The molecular weight of the enzyme is estimated at 200,000.     

Characterization of lysosomal acid lipase purified from rabbit liver

Lysosomal acid lipase from rabbit liver was solubilized with digitonin and purified 25,000-fold by Bio-Gel A-1.5 m, DEAE Bio-Gel A and phenyl Sepharose column chromatographies, preparative slab gel electrophoresis and finally Affi-Gel Blue affinity column chromatography. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis both in the presence and absence of sodium dodecyl sulfate. The molecular weight of the acid lipase was estimated to be 42,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 40,000 by gel filtration on Bio-Gel A-0.5 m. The enzyme was a hydrophobic glycoprotein with an isoelectric point of 5.15-5.90. The purified enzyme hydrolyzed tri-, di-, and monoolein and cholesterol oleate, with apparent Vmax values of 5.41, 56.1, 21.7, and 3.25 mumol/min/mg protein, and Km values of 50, 70, 200, and 40 microM, respectively. It hydrolyzed 4-methylumbelliferyl esters with fatty acids of different lengths in the order, medium length chains greater than long chains much greater than short chains. It did not hydrolyze dipalmitoylphosphatidylcholine. Its activity was inhibited by micromolar concentrations of p-chloromercuriphenyl sulfonic acid and p-bromophenacyl bromide and millimolar concentrations of Cu2+ and diethylpyrocarbonate. The activities of the enzyme towards the five substrates listed above showed almost identical thermal stabilities, mobilities on polyacrylamide gel electrophoresis and inhibition by several inhibitors. These findings support the idea that one enzyme is involved in the hydrolysis of both acylglycerols and cholesterol esters in lysosomes.     

Properties of NADPH-cytochrome P-450 reductase purified from rabbit liver microsomes.

NADPH-cytochrome P-450 reductase has been purified to electrophoretic homogeneity from rabbit liver microsomes by a procedure that may be used in conjunction with the isolation of the major forms of cytochrome P-450. The purified reductase is active in a reconstituted hydroxylation system containing P-450LM₂ or P-450LM₄. The enzyme contains one molecule each of FMN and FAD per polypeptide chain having an apparent minimal molecular weight of 74,000. Immunological techniques provided evidence for only a single form of the reductase; lower molecular weight forms occasionally seen are believed to be due to degradation by contaminating microsomal or bacterial proteases. Upon anaerobic photochemical reduction, the rabbit liver reductase undergoes spectral changes highly similar to those previously described by Vermilion and Coon for the rat liver enzyme; the fully reduced rabbit liver enzyme is converted to the three-electron-reduced form by the addition of NADP and then to the stable one-electron-reduced form by exposure to oxygen. The CD spectra of the fully oxidized enzyme, one-electron-reduced form (air-stable semiquinone), three-electron-reduced form, and fully reduced form are presented. The results obtained provide evidence that the FMN and FAD are in highly different environments in the enzyme, as also indicated by the different redox potentials and oxygen reactivities of the flavins.