Effect of polyamines on cyclic AMP-dependent and independent protein kinases from mouse epidermis.

Soluble extracts from mouse epidermis contained both cyclic AMP-dependent and independent protein kinases which could be separated by DEAE-Sephadex chromatography. The cyclic AMP-dependent histone kinase activity was inhibited by millimolar concentrations of the polyamines putrescine, spermidine and spermine. Similar concentrations of polyamines stimulated the cyclic AMP-independent phosphorylation of casein. The polyamines did not inhibit cyclic AMP binding by soluble epidermal extracts.     

Stimulation of valyl- and isoleucyl-tRNA synthetase reactions by polyamines

The aminoacylation of tRNA catalysed by valyl-tRNA synthetase (EC 6.1.1.9) and isoleucyl-tRNA synthetase (EC 6.1.1.5) fromMycobacterium smegmatis is dependent on the presence of divalent metal ions. Polyamines alone, in the absence of metal ions, do not bring about aminoacylation. In the presence of suboptimal concentrations of Mg²⁺, polyamines significantly stimulate the reaction. Of the cations tested, only Mn²⁺, Co²⁺ and Ca²⁺ can partially substitute for Mg²⁺ in aminoacylation, and spermine stimulates aminoacylation in the presence of these cations also. At neutral pH, spermine deacylates nonenzymatically aminoacyl tRNA. AMP and pyrophosphate-dependent enzymatic deacylation of aminoacyl-tRNA (reverse reaction) is also stimulated by spermine. The inhibitory effect of high concentration of KC1 on aminoacylation is counteracted, by spermine. The low level of activity between pH 8.5–9.0 at 1.2 mM Mg²⁺ is restored to normal level on the addition of spermine. The inhibitory effect of high pH on aminoacylation in the presence of low concentration of Mg²⁺ is also prevntedvby spemine.     

Differential effects of spermine on phosphatidylinositol 3-kinase and phosphatidylinositol phosphate 5-kinase

The metabolism of phosphoinositides plays an important role in the signal transduction pathways. We report here that naturally occuring polyamines affect the activities of phosphatidylinositol (PI) 3-kinase and PI 4-phosphate (PIP) 5-kinase differently. While polyamines inhibited the PI 3-kinase activity, they stimulated the activity of PIP 5-kinase in the order of spermine > spermidine > putrescine. Spermine inhibited the PI 3-kinase activity in a concentration-dependent manner with an IC₅₀ of 100 μM. On the other hand, spermine (5 mH) stimulated the activity of PIP 5-kinase 2–3 fold. Kinetic studies of spermine-mediated inhibition of PI 3-kinase revealed that it was noncompetitive with respect to ATP. The effect of Mg²⁺ and PIP, concentration on kinase activity was sigmoidal, with spermine inhibiting PI 3-kinase activity at all PIP₂ concentrations. While 1 mH calcium stimulated PI 3-kinase activity at submaximal concentrations of Mg²⁺ (1.25 mH), inhibition was observed at optimal concentration of Mg²⁺(2 mM). We propose that spermine may modulate the cellular signal by virtue of its differential effects on phosphoinositide kinases.     

Purification and characterization fo a phosvitin kinase from the thyroid gland

1. Two cyclic AMP independent protein kinases phosphorylating preferentially acidic substrates have been identified in soluble extract from human, rat and pig thyroid glands/ Both enzymes were retained on DEAE-cellulose. The first enzyme activity eluted between 60 and 100 mM phosphate (depending on the species), phosphorylated both casein and phosvitin and was retained on phosphocellulose; this enzyme likely corresponds to a casein kinase already described in many tissues. The second enzyme activity eluted from DEAE-cellulose at phosphate concentrations higher than 3000 mM, phosphorylated only phosvitin and was not retained on phophocellulose. These enzymes were neither stimulated by cyclic AMP, cyclic GMP and calcium, nor inhbiited by the inhibitor of the cyclic AMP dependent protein kinases. 2. The second enzyme activity was purified from pig thyroid gland by the association of affinity chromatography on insolubilized phosvitin and DEAE-cellulose chromatography. Its specific activity was increased by 8400. 3. The purified enzyme (phosvitin kinase) was analyzed for biochemical and enzymatic properties. Phosvitin kinase phosphorylated phosvitin with an apparent K_m of 100 μg/ml; casein, histone, protamine and bovine serum albumin were not phosphorylated. The enzyme utilized ATP as well as GTP as phosphate donor with an apparent K_m of 25 and 28 μM, respectively. It had an absolute requirement for Mg²⁺ with a maximal activity at 4 mM and exhibited an optimal activity at pH 7.0. The molecular weight of the native enzyme was 110 000 as determined by Sephacryl S300 gel filtration. The analysis by SDS-polyacrylamide gel electrophoresis revealed a major band with a molecualr weight of 35 000 suggesting a polymeric structure of the enzyme.     

Polyamine-like effects of cobalt(III) hexaammine on various cyclic nucleotide-independent protein phosphokinase reactions

The chemically inert trivalent ion cobalt(III) hexaammine, Co³⁺(NH₃)₆, was found to exert polyamine-like effects in enhancing certain cyclic nucleotide-independent protein kinase reactions catalyzed by nuclear enzyme preparations from rat ventral prostate or liver. At 1 mM, Co³⁺(NH₃)₆ stimulated chromatin- and also non-histone-protein-associated kinase activities with partially dephosphorylated phosvitin as substrate by 38% and 72% respectively, whereas chromatin-associated kinase-catalyzed phosphorylation of lysine-rich histones was not affected under the same conditions. ³²P incorporation (from γ-³²P-ATP) into endogenous protein substrates of chromatin or non-histone protein fractions catalyzed by their erdogencus kinase activity was increased by 47% and 153%, respectively. These effects of Co³⁺(NH₃)₆ were similar to those produced by 1mM spermine. Autoradiographic analysis of endogenous ³²P-labelled nonhistone proteins revealed similar enhancements of the phosphorylation of several of the same proteins, induced by 1mM spermine or 1 mM Co³⁺(NH₃)₆ or 2mM spermidine. The stimulatory actions of polyamines or Co³⁺(NH₃)₆ were not mimicked by raising the ionic strength by addition of comparable concentrations of NaCl. The effects of 1 mM spermine and of 1 mM Co³⁺(NH₃)₆ tested separately were not additive. Phosphorylation of lysine-rich histones by beef heart cyclic AMP-dependent protein kinase was not affected by polyamines or Co³⁺(NH₃)₆ Various findings hint that the enhancement of cyclic nucleotide-independent kinase-catalyzed phosphorylation of certain protein substrates by spermidine, spermine and Co³⁺(NH₃)₆ is primarily due to interaction of these cations with appropriate protein substrates affecting their conformational status. Further, these effects of polyamines may be a reflection of their cationic charge properties rather than being dependent on any particular conformations assumed by the polyamines.     

Spermine Stimulation of a Nuclear NII Kinase from Pea Plumules and Its Role in the Phosphorylation of a Nuclear Polypeptide

We have previously demonstrated that spermine stimulates the phosphorylation of a 47 kilodalton nuclear polypeptide from pea plumules (N Datta, LK Hardison, SJ Roux 1986 Plant Physiol 82: 681-684) In this paper we report that spermine stimulates the activity of a cyclic AMP independent casein kinase, partially purified from a chromatin fraction of pea plumule nuclei. This effect of spermine was substrate specific; i.e. with casein as substrate, spermine stimulated the kinase activity, and with phosvitin as substrate, spermine completely inhibited the activity. The stimulation by spermine of the casein kinase was, in part, due to the lowering of the Mg²⁺ requirement of the kinase. Heparin could partially inhibit this casein kinase activity and spermine completely overcame this inhibition. By further purification of the casein kinase extract on high performance liquid chromatography, we fractionated it into an NI and an NII kinase. Spermine stimulated the NII kinase by 5- to 6-fold but had no effect on the NI kinase. Using [γ-³²P]GTP, we have shown that spermine promotes the phosphorylation of the 47 kilodalton polypeptide(s) in isolated nuclei, at least in part by stimulating an NII kinase.     

Effects of cyclic adenosine 3': 5'-monophosphate on phosphoprotein kinase and phosphatase fractions prepared from rat liver nuclei.

A soluble rat liver nuclear extract containing total RNA polymerase activities also exhibits appreciable amounts of protein kinase activity. This unfractionated protein kinase catalyzes the phosphorylation of both endogenous proteins and exogenous lysine-rich histone in the presence of [γ-³²P]ATP and Mg²⁺. The optimal concentration of Mg²⁺ is 5 mm for histone phosphorylation and 25 mm for the phosphorylation of endogenous proteins. Cyclic AMP has no effect on the phosphorylation of lysine-rich histone by this unfractionated nuclear protein kinase. However, addition of cyclic AMP causes a reduction in the ³²P-labeling of an endogenous protein (CAI) which can be characterized by its mobility during SDS-acrylamide gel electrophoresis and elution in the unbound fraction of a DEAESephadex column. If CAI is first labeled with ³²P and then incubated with 10^?6m cyclic AMP under conditions where protein kinase activity is inhibited, the presence of the cyclic nucleotide causes a loss of the ³²P-labeling of this protein, implying the activation of a substrate-specific protein phosphatase. When rat liver RNA polymerases are purified by DEAE-Sephadex chromatography, protein kinase activity is found in the unbound fraction and in those column fractions containing RNA polymerase I and II. The fractionated protein kinases exhibit different responses to cyclic AMP, the unbound protein kinase being stimulated and the RNA polymerase-associated protein kinases being dramatically inhibited. A second protein (CAII) whose phosphorylated state is modified by cyclic AMP is found within the DEAE-Sephadex column fractions containing RNA polymerase II. The cyclic nucleotide in this case appears to reduce labeling of CAII by inhibition of the protein kinase activity which co-chromatographs with both CAII and RNA polymerase II. Based on molecular weight estimates, neither CAI nor CAII appears to be an RNA polymerase subunit. The identity of CAI as a protein factor whose phosphorylated state influences nuclear RNA synthesis is suggested by the fact that addition of fractions containing CAI to purified RNA polymerase II inhibits the activity of this enzyme, but only if CAI has been previously incubated in the presence of cyclic AMP.     

A plasmodium protein kinase that is developmentally regulated, stimulated by spermine, and inhibited by quercetin

Plasmodium berghei-infected murine red cells possess protein kinase activity that is associated with the isolated parasites. Schizonts contain significantly higher levels of this protein kinase than the more immature forms, suggesting a relationship between this enzyme activity and parasite development. Partially purified protein kinase has a Km for ATP of approximately 30 microMs, whereas the Km for GTP is approximately 300 microMs and the substrate preference is phosvitin greater than casein much greater than histone greater than protamine. The Mg2+ optimum is 10-20 mM, and the protein kinase activity is stimulated by the polyamines spermine and spermidine. The flavone, quercetin, inhibits the protein kinase activity in a competitive manner with respect to ATP (Ki approximately 3 microMs), and P chabaudi also has a very similarly regulated protein kinase. Protein kinases from both species are very similar to the type I casein kinase.     

Purification and characterization of a phosvitin kinase from the thyroid gland

1. Two cyclic AMP independent protein kinases phosphorylating preferentially acidic substrates have been identified in soluble extract from human, rat and pig thyroid glands. Both enzymes were retained on DEAE-cellulose. The first enzyme activity eluted between 60 and 100 mM phosphate (depending on the species), phosphorylated both casein and phosvitin and was retained on phosphocellulose; this enzyme likely corresponds to a casein kinase already described in many tissues. The second enzyme activity eluted from DEAE-cellulose at phosphate concentrations higher than 300 mM, phosphorylated only phosvitin and was not retained on phosphocellulose. These enzymes were neither stimulated by cyclic AMP, cyclic GMP and calcium, nor inhibited by the inhibitor of the cyclic AMP dependent protein kinases. 2. The second enzyme activity was purified from pig thyroid gland by the association of affinity chromatography on insolubilized phosvitin and DEAE-cellulose chromatography. Its specific activity was increased by 8400. 3. The purified enzyme (phosvitin kinase) was analyzed for biochemical and enzymatic properties. Phosvitin kinase phosphorylated phosvitin with an apparent Km of 100 micrograms/ml; casein, histone, protamine and bovine serum albumin were not phosphorylated. The enzyme utilized ATP as well as GTP as phosphate donor with an apparent Km of 25 and 28 microM, respectively. It had an absolute requirement for Mg2+ with a maximal activity at 4 mM and exhibited an optimal activity at pH 7.0. The molecular weight of the native enzyme was 110 000 as determined by Sephacryl S300 gel filtration. The analysis by SDS-polyacrylamide gel electrophoresis revealed a major band with a molecular weight of 35000 suggesting a polymeric structure of the enzyme.     

Manganese enhances the phosphorylation of membrane-associated proteins isolated from barley roots

Microsomal membranes isolated from barley roots (Hordeum vulgare L. cv. CM72) contained endogenous protein phosphorylation activities that were greatly enhanced by Mn²⁺. Mg²⁺ions also stimulated protein phosphorylation, but to a lesser extent than Mn²⁺. Ca²⁺ enhanced Mg²⁺, but not Mn²⁺-dependent phosphorylation. It is proposed that this strong enhancement by Mn²⁺ may be due to a greater affinity of Mn²⁺ than either Ca²⁺ or Mg²⁺ for both the Ca²⁺ and Mg²⁺ binding sites of certain kinases. Some Mn²⁺ stimulated kinase activity was eliminated from the membrane by washing with 0.2 mol/L KCl. The KCl extract contained histone and casein kinase activities, and 4 major phosphoproteins that were phosphorylated on serine and threonine residues. Phosphorylation of a 52 kDa polypeptide corresponded with the characteristics of the histone kinase activity and may represent the autophosphorylation of a CDPK-type kinase. Phosphorylation of a 36 kDa polypeptide was Ca²⁺ stimulated and may represent the autophosphorylation of a different type of unknown kinase. Polypeptides of 18 and 15 kDa had characteristics that suggest they were autophosphorylating subunits of a membrane bound nucleotide di-phosphokinase.     

Stimulation of the cyclic AMP-dependent protein kinase-catalyzed phosphorylation of phosphorylase kinase by micromolar concentrations of spermine

The phosphorylation of phosphorylase kinase by cyclic AMP-dependent protein kinase (A-kinase) is stimulated approximately 2-fold by spermine and spermidine. Half maximal effects were observed at 10 microM and 150 microM of spermine and spermidine, respectively. The phosphorylations of other substrates of A-kinase such as glycogen synthase, histone, and casein are not stimulated by these two polyamines. The rates, but not the final extents, of phosphorylation of both the alpha and beta subunits of phosphorylase kinase by A-kinase are stimulated by spermine. The results indicate that spermine and spermidine may play an important role in the activation of glycogenolysis in skeletal muscle.     

Purification and characterization of two cyclic AMP-independent casein/glycogen synthase kinases from rat liver cytosol

Two cyclic AMP-independent protein kinases (ATP: protein phosphotransferase, EC 2.7.1.37) (casein kinase 1 and 2) have been purified from rat liver cytosol by a method involving chromatography on phosphocellulose and casein-Sepharose 4B. Both kinases were essentially free of endogeneous protein substrates and capable of phosphorylating casein, phosvitin and I-form glycogen synthase, but were inactive on histone IIA, protamine and phosphorylase b. They were neither stimulated by cyclic AMP, Ca2+ and calmodulin, nor inhibited by the cyclic AMP-dependent protein kinase inhibitor protein. The casein and glycogen synthase kinase activities of each enzyme decreased at the same rate when incubated at 50 degrees C. Casein kinase 1 and casein kinase 2 showed differences in molecular weight, sensitivity to KCl, Km for casein and phosvitin and Ka for Mg2+, whereas their Km values for ATP and I-form glycogen synthase were similar. The phosphorylation of glycogen synthase by these kinases correlated with a decrease in the +/- glucose 6-phosphate activity ratio (independence ratio). However, casein kinase 1 catalyzed the incorporation of about 3.6 mol of 32P/85000 dalton subunit, decreasing the independence ratio from 83 to about 15, whereas the phosphorylation achieved by casein kinase 2 was only about 1.9 mol of 32P/850000 dalton subunit, decreasing the independence ratio to about 23. The independence ratio decrease was prevented by the presence of casein but was unaffected by phosphorylase b. These data indicate that casein/glycogen synthase kinases 1 and 2 are different from cyclic AMP-dependent protein kinase and phosphorylase kinase.     

Polyamines stimulate the activity of glycogen synthase (casein) kinase-1 from bovine kidney and different rat tissues

Previous studies have established that casein kinase-2 (CK-2) is stimulated by polyamines. In this study it is shown that glycogen synthase (casein) kinase-1 (CK-1) can be activated similarly. Using casein as the substrate, bovine kidney CK-1 was stimulated 7-, 2-, and 0.5-fold by spermine, spermidine, and putrescine, respectively. Half-maximal activation of CK-1 by these polyamines was observed at 0.25, 0.70, and 0.50 mM, respectively. CK-1 was optimally activated by spermine at low ionic strength and low Mg2+ concentrations (1-3 mM). Using phosvitin as the substrate, CK-1 was stimulated at low concentrations (0-0.8 mM) and inhibited at higher concentrations of spermine. By contrast CK-2 was inhibited at all concentrations of spermine when phosvitin was used as substrate. Using calcineurin (not a substrate for CK-2) as a substrate, CK-1 from bovine kidney or from three rat tissues (liver, kidney, and testis) was stimulated greater than 2-fold by spermine. It is further shown that heparin inhibits CK-1 and this inhibition can be reversed by spermine. The Vmax of CK-1 for both casein and ATP is increased by spermine while the Km remains unchanged by the polyamine. These studies indicate that CK-1, like CK-2, is a heparin-inhibited and polyamine-activated protein kinase. The results also suggest that CK-1 may be activated by spermine in vivo.     

Regulation of heart insulin receptor tyrosine kinase activity by magnesium and spermine

Insulin action and aspects of the insulin-signaling pathway have been studied in the heart although the direct regulation of the heart’s insulin receptor has not been explored. This study describes the first purification and characterization of the mammalian (rabbit, rat and bovine) heart insulin receptor. The rabbit heart IR showed maximum insulin binding of 18 μg/mg (~1 mole insulin/mole (α₂β₂) receptor) and a curvilinear Scatchard plot with a high affinity K_D for insulin binding of ~4 nM at optimal pH (7.8) and NaCl concentration (150 mM). The insulin receptor tyrosine kinase activity was stimulated by insulin, Mg²⁺ (half-maximum response at ~5.6–10.6 nM and ~8.5 mM, respectively) and by the physiological polyamines, spermine and spermidine. The stimulation by Mg²⁺ and the polyamines occurred with and without insulin. These characteristics of the heart insulin receptor provide a mechanism for regulating the activity of the receptor’s tyrosine kinase activity by the intracellular free Mg²⁺ concentration and the polyamines in the absence and presence of insulin.     

The effect of amino acids,monoamines and polyamines on pyruvate dehydrogenase activity in mitochondria from rat adipocytes

Summary The ability of polyamines and other cationic compounds including monoamines, amino acids, poly-L-arginine, poly-D-lysine and poly-L-lysine, to alter pyruvate dehydrogenase (PDH) activity in mitochondria from rat epididymal adipocytes was determined. PDH was assayed with the substrate [1-¹⁴C] pyruvate in the presence of 0.05 mM Ca²⁺ and Mg²⁺. Nine of the fourteen compounds tested at 0.1 mM caused a significant increase (procaine, 3-(-morpholinopropionyl) benzo[b]thiophene [VII], spermine, spermidine, putrescine, lysine and tryptophan) or decrease (poly-L-arginine, 3-(-piperidinopropionyl) benzo[b]thiophene) in PDH activity. None of these compounds nonenzymatically decarboxylated [1-¹⁴C] pyruvate to release ¹⁴CO₂. NaF, a PDH phosphatase inhibitor, suppressed the stimulatory effects of those compounds tested: procaine, tryptophan, VII, spermine and spermidine. These results imply that these five compounds activate PDH activity through stimulation of the PDH phosphatase. When the Mg²⁺ concentration was increased from 0.05 to 4.5 mM, the stimulatory effect of spermine was increased, consistent with the finding by others that spermine lowers the Km of the enzyme for Mg²⁺. However, at Mg²⁺ concentrations greater than 0.3 mM, the stimulatory effect of VII was unaltered, procaine failed to alter PDH activity, lysine inhibited PDH activity, and poly-L-lysine stimulated PDH activity. Therefore, polyamines and other positively charged small molecules may be physiologic regulators of PDH activity.     

Polyamine-sensitive protein kinase from chick intestinal mucosa

Summary A cyclic nucleotide-independent protein kinase which phoshorylates preferentially acidic proteins such as casein or phosvitin was isolated from cytosol of chick duodenal mucosa. The enzyme was purified more than 633 fold to apparent homogeneity by ammonium sulfate fractionation, column chromatography on DEAE-cellulose, phosphocellulose, hydroxylapatite and by sucrose density gradient centrifugation. The native enzyme has a molecular weight of 131000 as measured by gel filtration. The enzyme is a complex protein containing three polypeptides of molecular weight of 39 000, 36 000 and 27 000. It behaves as a complex throughout its purification and gel filtration but its components are readily separated by electrophoresis in denaturing buffer. The 27 000 molecular weight band was selectively autophosphorylated when the enzyme was incubated in the presence of [-³²P]ATP.When casein was used as substrate, physiological concentrations of naturally occurring polyamines such as spermine and spermidine markedly stimulated enzyme activity. However with phosvitin as substrate polyamines were strong inhibitors of the enzyme activity. This contrasting effect on intestinal kinase activity was also apparent using cytoplasmic proteins as endogenous phosphate acceptors. A characterization of this differential effect is presented and some possible physiological implications are discussed.     

The Ribosomal E Site at Low Mg2+: Coordinate Inactivation of Ribosomal Functions at Mg2+ Concentrations Below 10 mM and its Prevention by Polyamines

Abstract

Under standard conditions (Mg²⁺/150 mM NH₄ ⁺) ribosomes can quantitatively participate in tRNA binding at Mg²⁺ concentrations of 12 to 15 mM. The overall poly(U)-directed Phe incorporation and the extent of tRNA binding to either P,E or A sites decrease in a parallel manner when the Mg²⁺ concentration is lowered below 10 mM. At 4 mM the inactivation amounts to about 80%. The coordinate inactivation of all three binding sites is accompanied by an increasing impairment of the ability to translocate A-site bound AcPhe-tRNA to the P site. The translocation efficiency is already reduced at 10 mM Mg²⁺, and is completely blocked at 6—8 mM.

The severe inactivation seen at 6 mM Mg²⁺ vanishes when the polyamines spermine (0.6 mM) and spermidine (0.4 mM) are present in the assay; tRNA binding again becomes quantitative, the total Phe synthesis even exceeds that observed in the absence of polyamines by a factor of 4. In the presence of polyamines and low Mg²⁺ (3 and 6 mM) two essential features of the allosteric three-site model (Rheinberger and Nierhaus,J.Biol. Chem. 261, 9133 (1986)) are demonstrated. 1) Deacylated tRNA is not released from the P site, but moves to the E site during the course of translocation. 2) Occupation of the E site reduces the A site affinity and vice versa (allosteric interactions between E and A sites).

The quality of an in vitro system for protein synthesis can be assessed by two criteria. First, the incubation conditions must allow a near quantitative tRNA binding. Secondly, protein synthesis should proceed with near in vivo rate and accuracy. The 3 mM Mg²⁺/NH₄ ⁺/polyamine- system seems to be the best compromise at present between these two requirements.     

Partial Purification and Characterization of a Ca2+-Dependent Protein Kinase from Pea Nuclei

Almost all the Ca²⁺-dependent protein kinase activity in nuclei purified from etiolated pea (Pisum sativum, L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.3 molar NaCl. This protein kinase can be further purified 80,000-fold by salt fractionation and high performance liquid chromatography, after which it has a high specific activity of about 100 picomoles per minute per microgram in the presence of Ca²⁺ and reaches half-maximal activation at about 3 ×10⁻⁷ molar free Ca²⁺, without calmodulin. It is a monomer with a molecular weight near 90,000. It can efficiently use histone III-S, ribosomal S6 protein, and casein as artificial substrates, but it phosphorylates phosvitin only weakly. Its Ca²⁺-dependent kinase activity is half-maximally inhibited by 0.1 millimolar chlorpromazine, by 35 nanomolar K-252a and by 7 nanomolar staurosporine. It is insensitive to sphingosine, an inhibitor of protein kinase C, and to basic polypeptides that block other Ca²⁺-dependent protein kinases. It is not stimulated by exogenous phospholipids or fatty acids. In intact isolated pea nuclei it preferentially phosphorylates several chromatin-associated proteins, with the most phosphorylated protein band being near the same molecular weight (43,000) as a nuclear protein substrate whose phosphorylation has been reported to be stimulated by phytochrome in a calcium-dependent fashion.     

Lysine-rich histone H1 kinase from soybean hypocotyl.

A protein kinase (ATP: histone phosphotransferase) with high specificity for the phosphorylation of the very lysine-rich histone H1 has been partially purified and characterized from soybean hypocotyl. The enzyme has a molecular weight of about 48,500. Its activity and sedimentation behavior are refractory to cyclic nucleoside monophosphates. No significant amount of cyclic AMP or cyclic GMP binding activity could be detected in the crude or partially purified enzyme preparations. Km for ATP and histone H1 are 0.4 μM and 0.7 μM, respectively. The enzyme requires Mg²⁺ or Mn²⁺ for activity, while addition of 0.5 mM Ca²⁺, Zn²⁺ or Hg²⁺ results in 50% inhibition. Arginine-rich histones H3 and H4 are inhibitory to histone H1 phosphorylation; these histones affect the Vmax of the enzyme, but not the Km for histone H1.