Histone Phosphatase Activity Research Articles

Glycogen Synthetase-D Phosphatase: I. SOME NEW PROPERTIES OF THE PARTIALLY PURIFIED ENZYME FROM RABBIT SKELETAL MUSCLE

Abstract Purification of the phospho-protein phosphatase of skeletal muscle which promotes the conversion of the phospho- (D or b) form to the dephospho- (I or a) form of glycogen synthetase (UDPG:glycogen α-4-glucosyltransferase, EC 2.4.I.II) was aided by the use of buffers which include manganese chloride. The Mn2+ (5 mm) appeared to stabilize the enzyme and to insure greater recovery from DEAE-cellulose chromatography than reported previously. The enzyme was purified more than 1,000-fold from the 78,000 x g supernatant of rabbit muscle homogenate. It was essentially free of phosphorylase and synthetase. Phosphatase activity was measured by two methods: (a) as the rate of conversion of glycogen synthetase-D to the I form (D to I conversion) and (b) as the rate of orthophosphate release from 32P-labeled synthetase-D (32Pi release). Inhibition of the phosphatase reaction (more than 50%) was found in the presence of F- (10 mm), Na2SO3 (1 mm), and Pi or PPi (0.2 mm). More than a 2-fold increase in phosphatase activity was found in the presence of divalent metal cations: Mn2+ g Ca2+ g Mg2+ (half-maximal concentration was 0.6 to 1.2 mm). Glucose-6-P (up to 1 mm) had no effect on the rate of 32Pi release, but increased about 2-fold the rate of D to I conversion (at 0.1 mm), as did galactose-6-P and glucosamine-6-P to a lesser extent. A glycogen concentration of 0.5 to 1.5 mg per ml was found to be optimal compared to the slower reaction rates found with greater or lesser concentrations. A histone phosphatase activity found in this preparation, measured using 32P-labeled histone phosphate as substrate, had properties similar to and was copurified with glycogen synthetase-D phosphatase. However, the above carbohydrate-type effectors of synthetase and synthetase-D phosphatase reactions were without effect on the rate of histone dephosphorylation. These findings suggest that these two phosphatase activities may reside in the same enzyme protein.

Histone phosphatase and cyclic nucleotide-stimulated protein kinase from human lymphocytes.

1. Extracts of human peripheral blood lymphocytes contained a histone phosphatase that catalysed the release of P(i) from phosphorylated whole thymus histone. 2. Stimulation of the phosphatase was obtained by concentrations of KCl and NaCl of up to 75mm, and by MgCl(2); CaCl(2) inhibited the enzymic activity. 3. In the absence of MgCl(2), phosphoenol-pyruvate inhibited histone phosphatase activity; this inhibition could be partially reversed by adding MgCl(2) to assays. 4. Lymphocyte extracts contained a protein kinase activity which was maximally stimulated by 1mum-cyclic AMP (adenosine 3':5'-cyclic monophosphate) or by 0.1mm-cyclic GMP (guanosine 3':5'-cyclic monophosphate). 5. Incubation of the enzyme with histone in the absence of ATP or MgCl(2) resulted in the dissociation of the enzyme into a lower-molecular-weight species that was not stimulated by cyclic AMP. This effect could be prevented if ATP and MgCl(2) were present in reaction mixtures before histone and enzyme were allowed to interact. 6. Cyclic AMP also dissociated the kinase into a lower-molecular-weight species. 7. In the presence of 1mum-AMP, half-maximal activities were obtained with 0.92mm-MgCl(2), 6.0mum-ATP and 0.23mg of whole thymus histone/ml.

Changes in histone phosphorylation and associated early metabolic events in pig lymphocyte cultures transformed by phytohaemagglutinin or 6-N,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate.

1. Pig lymphocytes were transformed by dibutyryl cyclic AMP (6-N,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate) at concentrations of 0.01-0.1mum. The pattern of incorporation of label from [5-(3)H]uridine and [6-(3)H]thymidine into RNA and DNA respectively was identical with that obtained with unpurified phytohaemagglutinin. 2. Chlorpromazine (0.1mum) prevented the stimulation of [5-(3)H]uridine incorporation into RNA by phytohaemagglutinin, but only slightly lowered the lymphocyte response to dibutyryl cyclic AMP. 3. An increase in the size and specific radioactivity of the intracellular P(i) pool was found immediately after stimulation by both phytohaemagglutinin and dibutyryl cyclic AMP. This was followed after some 30min by a rise in the specific radioactivity and concentration of ATP. 4. There was an immediate increase in the specific radioactivity of phosphate groups of histones; by about 45min after stimulation only the histones remaining after extraction of histone fraction F1 continued to incorporate (32)P from [(32)P]P(i). 5. Histone kinase activity increased in the first 30min after stimulation; subsequently histone F1 kinase activity decreased, but activity with the other histones as substrate continued to increase for a further 30min. Kinase activation was effected by cyclic AMP (adenosine 3':5'-cyclic monophosphate). 6. Histone phosphatase activity behaved similarly to that of the kinase.

Histone phosphokinase activity in nuclear and cytoplasmic cell fractions from normal and regenerating rat livers.

1. Liver cell fractions were prepared by non-aqueous procedures and nuclei were also obtained in a hyperosmotic sucrose medium. Histone phosphokinase activity, assayed with histone F1 as substrate, was present in the soluble fraction of the cytoplasm and also bound on to the chromatin fraction of the nucleus. 2. The activity of the enzyme increased sixfold in nuclei from regenerating livers 22h after partial hepatectomy. 3. The enzyme bound in the nucleus was only marginally activated by 1mum-3':5'-cyclic AMP which stimulated the cytoplasmic soluble enzyme fourfold. 4. Nuclei prepared by the non-aqueous technique were also able to phosphorylate histones F2a and F3 and showed histone phosphatase activity with histone F1 phosphate as substrate.

Characterization of a Phosphatase Specific for Phosphorylated Histones and Protamine

Abstract A phosphatase which releases orthophosphate from histones and protamine has been purified 60-fold from rat liver. Histone phosphatase was detected by the use of substrates enzymatically phosphorylated by histone kinase. Purified histone phosphatase preparations are inactive in the dephosphorylation of other phosphoproteins, low molecular weight phosphate esters, and phosphopeptides derived from active substrates. The apparent Km for dephosphorylation of histone f1 is 2 x 10-5 m. The reaction products are orthophosphate and intact histone or protamine, as indicated by electrophoretic studies. Optimal reaction rates were observed at pH 7 to 8 and ionic strength 0.1 to 0.2. Chromatography on diethylaminoethyl cellulose resolved histone phosphatase activity into two fractions which exhibited a 2-fold difference in relative rates of dephosphorylation of histone fl and protamine. Histone phosphatase activity was detected in all eukaryotic cells examined, but it was absent in extracts of several prokaryotes.