Histone F2b Research Articles

The Type II Isoform of cGMP-dependent Protein Kinase Is Dimeric and Possesses Regulatory and Catalytic Properties Distinct from the Type I Isoforms

The type I cGMP-dependent protein kinases (cGK I alpha and I beta) form homodimers (subunit M(r) approximately 76,000), presumably through conserved, amino-terminal leucine zipper motifs. Type II cGMP-dependent protein kinase (cGK II) has been reported to be monomeric (M(r) approximately 86,000), but recent cloning and sequencing of mouse brain cGK II cDNA revealed a leucine zipper motif near its amino terminus. In the present study, recombinant mouse brain cGK II was expressed, purified, and characterized. Sucrose gradient centrifugation and gel filtration chromatography were used to determine M(r) values for holoenzymes of cGK I alpha (168,000) and cGK II (152,500), which suggest that both are dimers. Native cGK I alpha possessed significantly lower K alpha values for cGMP (8-fold) and beta-phenyl-1,N2-etheno-cGMP (300-fold) than did recombinant cGK II. Conversely, the Sp- and Rp-isomers of 8-(4-chloro-phenylthio)-guanosine-3',5'-cyclic monophosphorothioate demonstrated selectivity toward cGK II in assays of kinase activation or inhibition, respectively. A peptide substrate derived from histone f2B had a 20-fold greater Vmax/Km ratio for cGK I alpha than for cGK II, whereas a peptide based upon a cAMP response element binding protein phosphorylation site exhibited a greater Vmax/Km ratio for cGK II. Finally, gel filtration of extracts of mouse intestine partially resolved two cGK activities, one of which had properties similar to those demonstrated by recombinant cGK II. The combined results show that both cGK I and cGK II form homodimers but possess distinct cyclic nucleotide and substrate specificities.

Cyclic GMP-dependent protein kinase activation mediates inhibition of catecholamines secretion and Ca2+ influx in bovine chromaffin cells.

The effects of the membrane-permeable cGMP analogue, 8-bromoguanosine 3':5'-cyclic monophosphate on acetylcholine-evoked catecholamine secretion and cytosolic calcium increases were studied in chromaffin cells from the bovine adrenal gland. Preincubation with 100 microM 8-bromoguanosine 3':5'-cyclic monophosphate during 10 and 30 min decreased the acetylcholine-evoked catecholamine release by 16 +/- 3% and 27 /+- 5%, respectively. The cytosolic calcium increases triggered by acetylcholine and 30 mM KCl were also inhibited by 30 min of preincubation with this compound by 27 +/- 4 and 34 /+- 12%, respectively. Changes in membrane potential induced by acetylcholine and KCl were not affected by preincubation with 8-bromoguanosine 3':5'-cyclic monophosphate. The cyclic GMP-dependent protein kinase inhibitor N-[2-(methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride-at l micron abolished the inhibitory effect of 8-bromoguanosine 3':5'-cyclic monophosphate on acetylcholine-evoked calcium increase. By contrast, a potent and selective inhibitor against cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide did not block the 8-bromoguanosine 3':5'-cyclic monophosphate effect. Additionally, 8-bromoguanosine 3':5'-cyclic monophosphate stimulated histone F2b phosphorylation by a partial purified cGMP-dependent protein kinase from chromaffin cells. The extent of histone phosphorylation was reduced by N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride and 8-(4-chlorophenylthio)-guanosine 3':5'-cyclic monophosphorothioate, Rp-isomer, a specific inhibitor against cyclic GMP-dependent protein kinase, whereas it was not modified by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide. The results suggest that the inhibitory effects of 8-bromoguanosine 3':5' cyclic monophosphate on chromaffin cells are mediated through the activation of cGMP-dependent protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

High-affinity binding and localization of the cyclic GMP-dependent protein kinase with the intermediate filament protein vimentin.

The major receptor protein for cyclic GMP (cGMP) in smooth muscle is the cGMP-dependent protein kinase (cGMP kinase). The more abundant I alpha isoform (subunit M(r) congruent to 78,000) of this enzyme mediates the effects of cGMP to relax contracted vascular smooth muscle preparations. In this study, we have addressed the hypothesis that the cGMP kinase is anchored to intracellular proteins which might serve to target cGMP kinase to protein substrates. Using a gel overlay technique, immunoprecipitation, and a fluorescence binding assay for cGMP kinase, we have identified vimentin as a high-affinity and specific binding protein for cGMP kinase. Binding of cGMP kinase to vimentin is reversible and stoichiometric (one cGMP kinase dimer/vimentin dimer) with a KD of approximately 49 nM. The site of high-affinity binding between cGMP kinase and vimentin did not appear to be localized to the catalytic domain of the kinase since vimentin phosphorylated by cGMP kinase and peptide substrates for cGMP kinase did not compete for high-affinity binding. Neither the proteolytically-derived 69-kDa catalytic fragment nor the 8-kDa N-terminal fragment bound vimentin with high affinity, suggesting that the cGMP kinase dimer was necessary for the interaction. Vimentin was readily phosphorylated in vitro with the dimer, but not the monomeric 69-kDa catalytic fragment even though the monomeric 69-kDa fragment was catalytically active toward other substrates such as histone F2b and peptides. This suggests that the high-affinity interaction between cGMP kinase and vimentin occurs at the N-terminal region, thus allowing the interaction between the phosphorylation site of vimentin and the catalytic site of cGMP kinase to occur.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic GMP-Dependent Protein Kinase Activity in Rat Pulmonary Microvascular Endothelial Cells

Cyclic GMP-dependent protein kinase (cGPK) activity was determined in rat pulmonary microvascular endothelial cells (RPMVEC) using cGMP-stimulated phosphorylation of BPDEtide and histone F2B substrates in the presence of PKI [peptide inhibitor of cAMP-dependent protein kinase (cAPK)]. RPMVEC cGPK activity was localized to the 100,000 × g cytosolic fraction. The EC50 for cGMP activation in the presence of PKI was 0.16 μM and H-89 inhibition under similar conditions showed an IC50 value of 0.16 μM. Anion-exchange chromatography of RPMVEC and rat lung cytosolic fractions showed separation of the cGMP-dependent from the cGMP-independent protein kinase activity and similar elution conductivities. Further, Western blots of RPMVEC active DEAE-Trisacryl fractions showed immunoreactivity using bovine Type I cGPK antiserum. Preliminary studies reveal six potential substrates phosphorylated by cGPK in RPMVEC. These studies describe an endothelial cell (EC) cGMP-receptor, cGPK, in addition to cGMP-activated (Type II) phosphodiesterase (PDE).

Polypeptide hormone receptor phosphorylation: is there a role in receptor-mediated endocytosis of human growth hormone?

To determine whether receptor phosphorylation is a critical step in the internalization of polypeptide hormones and their receptors, we have studied a model system wherein insulin stimulates phosphorylation of its receptor and is also internalized. Using insulin as a positive control, we found that it stimulated a partially purified plasma membrane preparation of IM-9 lymphocytes to autophosphorylate its receptor and to catalyze the phosphorylation of a tyrosine-containing substrate. The human GH (hGH) receptor of the IM-9 lymphocytes, when coupled to [125I]iodo-hGH, migrated as a 140,000-dalton protein on polyacrylamide gel electrophoresis. This protein, in contrast to the insulin receptor, was not phosphorylated by the addition of hGH, nor did hGH stimulate this preparation to phosphorylate the tyrosine-containing substrate poly-(GluNa,Tyr)4:1, casein, or histone f2b under a variety of conditions. We conclude that receptor phosphorylation is not a critical intermediate in the receptor-mediated endocytosis of hGH and probably other polypeptide hormones and growth factors.

Analysis of hormone- and polynucleotide/histone-binding sites of the chicken intestinal 1,25-dihydroxyvitamin D3 receptor by means of proteolysis.

Several discrete forms of the 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor from chicken intestinal cytosol were characterized by ultracentrifugation; gel filtration; DNA-, histone-, dye-ligand-binding affinity; and several other chromatographic media. Formation of altered receptor complexes was carried out through partial proteolysis using trypsin, alpha-chymotrypsin, and papain. The holoreceptor complex was found to be asymmetric and have a Stoke's radius of 37 A. Depending on the concentration of protease, several sterol-binding fragments of 33 and 27 A were produced which tended to be more globular in shape. The 27 A complex was incapable of binding DNA, histones, or phosphocellulose, but still retained affinity for DEAE-cellulose. Resolution of three forms of 1,25-(OH)2D3 receptors was demonstrated by cibacron blue F3GA-agarose chromatography. The 37 A complex eluted at 1.1 M KCl, and the 33 A form eluted at 0.6 M KCl, whereas the 27 A complex was not retained by the column and eluted in the 0.15 M KCl wash. The specificity of the 37 A complex for histone binding was assessed. The order of binding preference was: histone f3 greater than histone f2a approximately equal to histone f2b much greater than histone f1. Results also indicated that DNA could inhibit receptor binding to histone and that this was competitive with respect to histone-agarose binding, suggesting that the interaction of histones and DNA is at a domain common to polynucleotides. It is concluded the 1,25-(OH)2D3 receptor has separate and distinct binding sites for hormone and polynucleotides/histones. These in vitro findings of histone binding suggest that the polynucleotide domain of this receptor is capable of recognizing several nuclear derived components that may be important for the alteration of gene expression.

Insulin-induced increases in the activity of the spontaneously active and ATP.Mg-dependent forms of phosphatase-1 in alloxan-diabetic rat liver.

Liver supernatant from normal and alloxan-diabetic rats was fractionated by DEAE-cellulose chromatography and the separated phosphoprotein phosphatase fractions were assayed with [32P]histone f2b, [32P]phosphorylase a and [32P]phosphorylase kinase as substrates. In diabetic rat liver, one of the phosphatase fractions found in the normal liver was significantly reduced. This fraction was identified as a mixture of the spontaneously active form and the ATP . Mg-dependent form of phosphoprotein phosphatase-1 (Fc) based on sensitivity to inhibitor-2, substrate specificity, and the fact that it could be activated 42-70% by glycogen synthase kinase-3 in the presence of ATP . Mg. Further analysis of this fraction showed that liver cytosol from diabetic rats contained 62-79% lower spontaneously active phosphatase-1 activity and 40-51% lower combined spontaneously active and ATP . Mg-dependent protein phosphatase-1 (Fc) activity. Insulin administration increased the spontaneously active and the ATP . Mg-dependent protein phosphatase-1 activities approximately 45% and 36%, respectively, in alloxan-diabetic rats. These data imply that the lower levels of spontaneously active phosphatase-1 activity in diabetic rat liver cannot be explained by presuming phosphatase-1 to have been present as Fc, the inactive form. Moreover, insulin restored the total activity of the spontaneously active and activatable forms of phosphatase-1 to those present in normal liver implying that both forms of phosphatase-1 activity are under hormonal control.

Calmodulin-dependent protein kinase and associated substrates in Torpedo electric organ.

Calcium plus calmodulin (Ca2+/CaM)-dependent protein kinase activity was demonstrated in subcellular fractions from Torpedo californica electric organ. A protein kinase activity dependent on Ca2+/CaM was purified about 200-fold from electric organ cytosol using DEAE-cellulose and CaM-affinity chromatography. The most effective exogenous substrates for this enzyme were the synapse-specific protein Synapsin I (Protein I) and histone f3. Phosphorylase b, skeletal muscle myosin light chains, casein, phosvitin, histone f2b, and G-substrate were relatively poorly phosphorylated by Torpedo CaM-protein kinase. Thus, the enzyme differs in its substrate specificity from known cyclic nucleotide-dependent protein kinases, myosin light chain kinase and phosphorylase kinase. The Km for ATP was 15-20 microM; for Synapsin I, 0.8 microM; and for CaM, 85 nM. Two major endogenous substrates (Mr = 62,000 and 54,000) for CaM-protein kinase co-purified with the enzyme through the CaM-affinity column step. These two substrates, as well as the enzyme, were present in other subcellular fractions in addition to the cytosol, including crude membranes and purified synaptic vesicles. A third major substrate (Mr = 39,000) could be separated from the enzyme during purification and appeared to be localized primarily in the cytosol. CaM-protein kinase increased the phosphorylation of both serine and threonine residues in endogenous substrates. In contrast to previous reports, no evidence for Ca2+/CaM-dependent phosphorylation of any subunit of the acetylcholine receptor was obtained.

CAMP-dependent protein kinase phosphorylates the nicotinic acetylcholine receptor.

Postsynaptic membranes, rich in the nicotinic acetylcholine receptor, were isolated from the electric organ of Torpedo californica and shown to contain a cAMP-dependent protein kinase and a calcium/calmodulin-dependent protein kinase. The cAMP-dependent protein kinase phosphorylated the gamma and delta subunits of the acetylcholine receptor. The phosphorylated subunits were identified after purification of the acetylcholine receptor by affinity chromatography on a choline carboxymethyl affinity gel. In contrast, the calcium/calmodulin-dependent protein kinase phosphorylated proteins that were separated from the acetylcholine receptor by affinity chromatography. Protein kinase inhibitor, a specific inhibitor of the catalytic subunit of cAMP-dependent protein kinase, abolished the basal endogenous phosphorylation of the gamma and delta subunits of the receptor. cAMP activation of the endogenous phosphorylation of the gamma and delta subunits was dose dependent with a half-maximal response at 25 nM. Studies were also carried out with acetylcholine receptor purified from T. californica and catalytic subunit of cAMP-dependent protein kinase purified from bovine heart. The purified acetylcholine receptor was rapidly and specifically phosphorylated on the gamma and delta subunits by the purified catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 1.0 and 0.89 mol of (32)P per mol of receptor, respectively. The initial rates of phosphorylation of the gamma and delta subunits of the receptor were comparable to those of histone f2B and synapsin I (protein I), two of the most effective substrates for the catalytic subunit. Under the conditions used, the gamma and delta subunits had K(m) values of 4.0 and 3.3 muM and V(max) values of 2.7 and 2.1 mumol/min per mg, respectively. The results are consistent with the idea that the acetylcholine receptor is phosphorylated in vivo by a cAMP-dependent protein kinase.

Cyclic AMP-dependent protein kinase activity and endogenous protein phosphorylation in isolated cortical segments of rabbit nephron.

Cyclic AMP-dependent protein kinase activity and endogenous protein phosphorylating activity are reported for 6 cortical segments of the rabbit nephron which were microdissected and collected according to their morphology. The 6 cortical nephron segments, namely the glomerulus (Glm), proximal convoluted tubule (PCT), proximal straight tubule (PST), cortical ascending limb of Henle's loop (CAL), distal convoluted tubule (DCT), and cortical collecting tubule (CCT), showed protein kinase activities which were increased 1.8-4.9 fold by 10(-6) M cyclic AMP in the presence of histone IIA, histone f2b or histone f3 as a protein substrate. However, all these segments showed little or no cyclic AMP dependent increase of activity with either protamine or alpha-casein as a protein substrate. Cyclic AMP increased the endogenous protein phosphorylation of Glm (10(-6) M cyclic AMP), of CAL (10(-7) and 10(-8) M cyclic AMP), of DCT (10(-6) M cyclic AMP) and of CCT (10(-8), 10(-7) and 10(-6) M cyclic AMP). In contrast, PCT showed decreased endogenous protein phosphorylation in the presence of 10(-7), 10(-5) and 10(-4) M cyclic AMP.

CYCLIC AMP-DEPENDENT PROTEIN KINASE ACTIVITY AND ENDOGENOUS PROTEIN PHOSPHORYLATION IN ISOLATED CORTICAL SEGMENTS OF RABBIT NEPHRON

Cyclic AMP-dependent protein kinase activity and endogenous protein phosphorylating activity are reported for 6 cortical segments of the rabbit nephron which were microdissected and collected according to their morphology. The 6 cortical nephron segments, namely the glomerulus (Glm), proximal convoluted tubule (PCT), proximal straight tubule (PST), cortical ascending limb of Henle’s loop (CAL), distal convoluted tubule (OCT), and cortical collecting tubule (CCT), showed protein kinase activities which were increased 1.8-4,9 fold by 10-6 M cyclic AMP in the presence of histone MA, histone f2b or histone f3 as a protein substrate. However, all these segments showed little or no cyclic AMP dependent increase of activity with either protamine or α-casein as a protein substrate. Cyclic AMP increased the endogenous protein phosphorylation of Glm (10-6 M cyclic AMP), of CAL (10-7 and 10-8 M cyclic AMP), of DCT (10-6 M cyclic AMP) and of CCT (10-5, 10-7 and 10-6 M cyclic AMP). In contrast. PCT showed decreased endogenous protein phosphorylation in the presence of 10-7,10-5 and 10-4 M cyclic AMP.

Differences in the Cyclic AMP-Dependent Phosphorylation of Plasma Membrane Proteins of Differentiated and Undifferentiated L 6 Myogenic Cells

Differences in the cyclic AMP-dependent plasma membrane phosphorylation system of undifferentiated and differentiated L6 myogenic cells have been detected. Endogenous plasma membrane protein phosphorylation in undifferentiated L6 myoblasts was stimulated more than three fold by 5 x 10(-5) M cyclic AMP, whereas no statistically significant cyclic AMP-dependent phosphorylation of endogenous plasma membrane proteins was observed in differentiated L6 cells. In undifferentiated cells cyclic AMP promoted the phosphorylation of several proteins, the most prominent of which had a molecular weight of 110,000. In differentiated cells cyclic AMP did not selectively promote the phosphorylation of specific plasma membrane proteins. Both differentiated and undifferentiated L6 cells, however, contain a cyclic AMP-dependent protein kinase capable of catalyzing the phosphorylation of exogenous substrates, such as histone f2b. Therefore, the data show that differentiation in L6 cells is associated with a selective change in the activity of a plasma membrane cyclic AMP-dependent protein kinase which employs endogenous membrane proteins as substrate.

Immunological distinction between guanosine 3':5'-monophosphate-dependent and adenosine 3':5'-monophosphate-dependent protein kinases.

A guanosine 3':5'-monophosphate (cGMP)-dependent protein kinase was purified from bovine lung using 8-(6-aminohexylamino)-cAMP-Sepharose. The activity of the purified enzyme was highly dependent on cGMP using histone f2b as a substrate. The self-phosphorylation of the purified enzyme was strongly inhibited by cGMP and not significantly affected by cAMP. A precipitating antiserum prepared in rabbits against the cGMP-dependent protein kinase specifically inhibited the histone kinase activity and the self-phosphorylation of the purified cGMP-dependent protein kinase without affecting the cGMP binding site. This antiserum also specifically inhibited the phosphorylation of the endogenous substrate proteins by endogenous cGMP-dependent protein kinase in smooth muscle membranes, but did not cross-react detectably with catalytic subunit or regulatory subunit of type I or type II cAMP-dependent protein kinase. Conversely, anti-sera against the regulatory subunit of type I or type II cAMP-dependent protein kinase did not cross-react detectably with cGMP-dependent protein kinase. The substantial differences between the immunological properties of the cGMP-dependent and cAMP-dependent protein kinases suggest that these two enzymes have distinct physiological roles.

Protein phosphatase from rat liver nuclei.

Protein phosphatase, active on non-histone phosphoprotein substrate, was partially purified from rat liver cell nuclei by means of salt extraction, ammoniumsulfate precipitation, DEAE cellulose chromatography, gel filtration and preparative isoelectrofocusing. Rat liver nuclei contain a heterogenous population of different protein phosphatases. All the enzyme fractions eluted from DEAE cellulose are of low molecular weight between 12,000--31,000. The pH 5.5 peak fraction of preparative isoelectrofocusing was characterized in detail. It has a pH optimum of 6.8 using nuclear phosphoprotein substrate. It is inhibited by Na+ at 80 mM, and to a lesser extent by K+, activated by Mg2+ (5 mM) and Mn2+ (1 mM). However, the latter is inhibitory at 6 mM. The nuclear protein phosphatase is also active on labelled F1 and F2b histones and casein, however, its V is lower on histones and it contains component(s) active specifically on nuclear phosphoprotein substrate but not on casein.

The in vivo distribution of immunoreactive larger than tetrameric polyadenosine diphosphoribose in histone and non-histone protein fractions of rat liver.

The macromolecular association of immunoreactive naturally occurring polyadenosine diphosphoribose n greater than 4 with histones and non-histone proteins was determined with the aid of an improved method of extraction of polyadenosine diphosphoribose and a combination of radioimmunoassay and molecular filtration. More than 99% of the naturally occurring polyadenosine diphosphoribose n greater than 4 was present in rat liver in covalent association with non-histone proteins. The chain length of the polymer varied between n = 4 and n = 34. Less than 1% of naturally occurring polyadenosine diphosphoribose n greater than 4 was almost evenly distributed between histone fractions f1, f2a, f2b, and f3. Adenosine diphosphoribose polymers of relatively long chain length were also detected in the histone fractions. The covalent association of polyadenosine diphosphoribose with non-histone proteins was demonstrated by affinity chromatography.

Effects of some carcinogens on conformation of histones

AbstractConformational transitions of histones F1 (H1), F2a (H2A), F2b (H2B), and F3 (H3) in the presence of carcinogens 4‐nitroquinoline‐N‐oxide, 13‐H‐dibenzo[c,j]carbazole, and 7‐H‐dibenzo[c,q]carbazole were studied by the circular dichroism (CD) probe. The polypeptide main chain conformation of the very lysine rich histone F1 was not affected by these carcinogens, but the tertiary structure was modified slightly by the carbazoles. The secondary structure of histone F2a was significantly modified by the carbazoles which enhanced the helix content, whereas the effect of the 4‐nitroquinoline‐N‐oxide on the backbone conformation was weak. The tertiary structure of F2a was affected by all three carcinogens, whereby the 7‐H‐dibenzo[c,g]carbazole produced the strongest enhancement of the CD bands assigned to tyrosine and phenylalanine chromophores. Moreover, this carcinogen produced extrinsic Cotton effects in the 300–380nm spectral zone. Similar effects of the carbazoles were observed with histone F2b, although in this case the main chain conformation was changed only slightly. The secondary structure of histone F3 was slightly affected by the carcinogens but the tertiary structure was significantly modified by the carbazoles. The CD bands of F3 which were assigned to the tyrosine and phenylalanine chromophores were most strongly enhanced by the 13‐H‐dibenzo[c,j]carbazole.

Replacement of protamine by F1 histone during reactivation of fused human sperm nuclei

Rabbit antisera, specific for the histones F1, F2a2, F2b, F3 and for protamine were used to monitor a possible transition from protamine towards somatic-type histones during sperm nucleus reactivation, following human sperm fusion with mouse fibroblasts. Mature human sperm nuclei were shown to contain the histones F2a2, F2b, F3 and protamine, but were missing F1 histone by immuno cytochemistry using the indirect fluorescence method. However, a gradual disappearance of protamine from fused sperm nuclei, could be observed during the first 24 h of reactivation. Subsequently, F1 histone could be detected in increasing concentrations in 60% of reactivated sperm nuclei, during the next four days. The shift from protamine towards F1 histone could also be visualized cytochemically via staining with brilliant sulphaflavine, which appears to discriminate between sperm nuclei on the basis of their F1 histone content.

Action of estrogens on reconstituted nucleohistones

The action of estrone, 17β-estradiol, and estriol on the degree of binding of histones with DNA in reconstituted nucleohistones and the effect of these hormones on thermal denaturation of nucleoproteins were studied. Estrogens in concentrations of 10−5 M were shown not to change the protein/DNA ratio by weight in partially reconstituted F1, F2a, F2b, and F3 nucleohistones and in total nucleohistone reconstituted from DNA and an equimolar complex of histones F2a, F2b, and F3. Treatment with estrogens in the above-mentioned concentration does not change the melting point of any of the nucleohistones studied. The effect of estrogens on thermal denaturation of total nucleohistone and of partially reconstituted nucleohistones F2a, F2b, and F3 consists of an increase in the hyperchromic effect of the first phase of melting and a corresponding decrease in the hyperchromic effect of the other phases. Estrogens did not change the thermal denaturation profile of partially reconstituted nucleohistone F1.

Some properties of the cyclic AMP dependent protein kinase of epithelial cells and cortical fibers of bovine eye lens

Cyclic AMP dependent protein kinase activity was demonstrated in extracts of epithelial cells and cortical fibers of bovine eye lens. The histone fractions can be phosphorylated by these enzymes. Highest phosphorylation was obtained with f2b and f1 histone fractions. The K m values of the epithelial and cortical enzymes were very similar with all histone fractions. Protein kinase in the two extracts had a similar affinity to ATP and showed a pH optimum at 6·0–6·2. The enzymes were activated by cyclic AMP and the concentration of cyclic AMP necessary to half maximal activation was similar for both enzymes: 5×10 −8 m for the epithelial enzyme and 9×10 −8 m for the cortical enzyme. The magnitude of activation, however, was very different. The activity of the epithelial enzyme was increased 5–8-fold, while that of the cortical enzyme up to 25-fold. The high cyclic AMP sensitivity of the cortical enzyme was accompanied by a relatively high cyclic AMP binding capacity.

Studies of Interaction between Histone F2b and DNA from Normal and Exposed to X-radiation Calf Lymph Nodes

Studies of Interaction between Histone F2b and DNA from Normal and Exposed to X-radiation Calf Lymph Nodes