Energy-liked reactions in photosynthetic bacteria. X. Solubilization of the membrane-bound energy-linked inorganic pyrophosphatase of Rhodospirillum rubrum.

The energy-linked membrane-bound inorganic pyrophosphatase of $\text{Rhodospirollum}$$\text{rubrum}$, G-9, has been solubilized with good yield from chromatophores using cholate in the presence of MgCl₂. The enzyme has been partially purified using ammonium sulfate fractionation and gel chromatography. After fractionation the enzyme requires phospholipid for activity. The solubilized enzyme is specific for PP_i and requires Mg²⁺ for activity as has been found for other PP_iases.     

Solubilization and partial purification of cytochrome P-450 from rat lung microsomes

Cytochrome P-450 from rat lung microsomes has been solubilized and purified 8-fold by using affinity chromatography on an ω-amino-$\text{n}$-octyl derivative of Sepharose 4B. The purified fraction was free of cytochrome $\text{b}$₅ and NADPH-cytochrome $\text{c}$ reductase and showed spectral characteristics similar to those of lung microsomal cytochrome P-450. When combined with NADPH-cytochrome $\text{c}$ reductase partially purified from liver microsomes, the cytochrome P-450 fraction supported the hydroxylation of benzo (α)pyrene and the activity was proportional to the content of the hemoprotein. No absolute requirement for phosphatidylcholine was found.     

Characteristics of a nuclear protein kinase from rat epididymis

Purified rat epididymal nuclei possess a cyclic AMP-independent protein kinase activity that phosphorylates of casein. The enzymic activity was solubilized by treating intact nuclei with 1 M (NH4)₂SO₄. One major peak of kinase activity was obtained when the solubilized enzyme preparation was subjected to diethylaminoethyl-Sephadex chromatography. The activity of the kinase was dependent on a bivalent metal ion such as Mg²⁺, Co²⁺, Ca²⁺ or Mn²⁺. NaCl (0.3 M) caused a further activation (approx. 200%) of the metal (Co²⁺)-dependent enzyme. The apparentK _m values of the enzyme for casein, ATP and Co²⁺ are approx. 0.6 mg/ml, 10 ΜM and 2.2 mM respectively. The enzyme was maximally active at pH 5.5. The enzyme showed high specificity for phosphorylation of the acidic protein casein but did not phosphorylate basic proteins, such as histones and protamine. The properties of the nuclear protein kinase were clearly different from those of the cytosolic enzymes previously characterized.     

Phosphorylation of calf uterus 17 beta-estradiol receptor by endogenous Ca2+-stimulated kinase activating the hormone binding of the receptor

Direct evidence is presented that uterus 17_β-estradiol receptor is phosphorylated $\text{in}\text{,}\text{vitro}$ by an endogenous kinase. Nuclear phosphatase, cytosol Ca²⁺-stimulated kinase (the former inactivating and the latter reactivating the hormone binding of the 17_β-estradiol receptor) and receptor were purified from calf uterus. 17_β-estradiol binding was inactivated by phosphatase, then reactivated by kinase in the presence of [_γ-³²P] ATP, Ca²⁺ and calmodulin, and the receptor was examined by various methods. The results of gel electrophoresis in non denaturating and denaturating conditions, and of centrifugation through sucrose gradients of receptor preincubated with monoclonal antibodies showed that the receptor is phosphorylated.     

Properties and partial purification of mevalonate kinase from Agave americana

Mevalonate kinase activity was demonstrated in acetone powder extracts from Agave americana leaves, flowers and scape. ATP was the most effective phosphate donor. The enzyme had an optimum pH of 7.9 in Tris-HCl buffer. Dialysis decreased the ability to phosphorylate mevalonic acid (MVA). Partially purified mevalonate kinase reached maximum activity in the presence of 2 mM Mn²⁺ or 6–8 mM Mg²⁺. Higher concentrations of Mn²⁺ were inhibitory, whereas higher concentrations of Mg²⁺ produced only a small decrease in the activity. The amount of mevalonate-5-phosphate (MVAP) formed depended on protein concentration and incubation time. During short incubations, the MVAP formed increased as protein concentration rose, whereas during prolonged incubations (1–6 hr), there was a decrease in the MVAP formed when a certain amount of protein was exceeded. It is suggested that MVAP formed was hydrolysed by a phosphatase present in the extracts. This interfering activity was eliminated when mevalonate kinase is partially purified. The apparent K_m values of the enzyme from leaves were 0.05 mM for MVA and 0. 14 mM for ATP. Similar K_m values are obtained with partially purified mevalonate kinase. The enzyme was purified by ammonium sulphate precipitation, Sephadex G-100 filtration and DEAE-Sephadex A-50 fractionation.     

TYROSINE HYDROXYLASE IN BOVINE CAUDATE NUCLEUS

Approximately 80 per cent of tyrosine hydroxylase activity in bovine caudate nucleus was particle-bound. The rest of the activity was found in the soluble fraction. The enzyme activity in crude tissue preparations was inhibited, probably by the presence of endogenous inhibitors. Dilution of crude tissue preparations such as the crude mitochondrial fraction caused an increase in the specific activity. The particle-bound enzyme was solubilized by incubation with trypsin. The presence of deoxycholate increased the degree of solubilization. The activity of the solubilized enzyme from the washed particles was also inhibited, but the subsequent purification by ammonium sulphate could eliminate the inhibition. The solubilized enzyme was partially purified by ammonium sulphate fractionation and Sephadex G-150 chromatography. A tetrahydropteridine and ferrous ion were required as cofactors for the partially purified enzyme. Among various divalent cations, only ferrous ion could activate the partially purified enzyme. The enzyme was inhibited by L-α-methyl-p-tyrosine and catecholamines such as dopamine. The optimum pH was found between 5.5 and 6.0. K_m values toward tyrosine, 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine and Fe²⁺, were approximately 5 × 10^?5 M, 1 × 10^?4 M and 4 × 10^?4 M, respectively.     

Terminal deoxynucleotidyl transferase: characterization of extraction and assay conditions from human and calf tissue.

Methods of extraction and assay of terminal deoxynucleotidyl transferase (TdT) from human lymphoblasts and calf thymus were compared. A high salt concentration was mandatory for complete enzyme extraction, while dialysis of the crude extract resulted in a major loss of enzyme activity. In addition, TdT was partially purified from lymphoblasts of patients with acute lymphoblastic leukemia. The K_m for the monomer, deoxy-guanosine 5′-triphosphate (dGTP), is high (~0.1 mm) in the presence of either Mg²⁺ or Mn²⁺, whereas the K_m for the initiator, poly(deoxyadenylic acid $\text{[poly(d(pA)}\text{50}\text{)]}$, with an average chain length of 50 residues, is 2.5 μm in the presence of Mg²⁺ and 0.3 μm in the presence of Mn²⁺. The maximum velocity is higher for the calf thymus TdT in the presence of Mg²⁺ than in Mn²⁺. Human TdT catalyzes the polymerization of dGTP at a higher rate in the presence of Mn²⁺ than with Mg²⁺. These data illustrate that partially purified human TdT differs in catalytic properties from the purified calf thymus enzyme. Therefore, optimal conditions for assay of TdT in extracts from calf and human tissues differ.     

The association of phosphorylase kinase with rabbit muscle T-tubules

Evidence is presented for the association of a phosphorylase kinase activity with transverse tubules as well as terminal cisternae in triads isolated from rabbit skeletal muscle. This activity remained associated with T-tubules throughout the purification of triad junctions by one cycle of dissociation and reassociation. The possibility that the presence of phosphorylase kinase in these highly purified membrane vesicle preparations was due to its association with glycogen was eliminated by digestion of the latter with α-amylase. The phosphorylase kinase activity associated with the T-tubule membranes was similar to that reported for other membrane-bound phosphorylase kinases. The enzyme had a high $\text{pH}\text{6.8}\text{pH}\text{8.2}$ activity ratio (0.4 – 0.7) and a high level of Ca²⁺ independent activity $\text{(EGTA}\text{Ca}{}^{\mathrm{2+}}\text{= 0.3?0.5)}$. The kinase activated and phosphorylated exogenous phosphorylase b with identical time courses. When mechanically disrupted triads were centrifuged on continuous sucrose gradients, the distribution of phosphorylase kinase activity was correlated with the distribution of a M_r 128,000 polypeptide in the gradients. This polypeptide and a M_r 143,000 polypeptide were labeled with ³²P by endogenous and exogenous protein kinases. These findings suggest that the membrane-associated phosphorylase kinase may be similar to the cytosolic enzyme. Markers employed for the isolated organelles included a M_r 102,000 membrane polypeptide which followed the distribution of Ca²⁺-stimulated 3-O-methylfluorescein phosphatase activity, which is specific for the sarcoplasmic reticulum. A M_r 72,000 polypeptide was confirmed to be a T-tubule-specific protein. Several proteins of the triad component organelle were phosphorylated by the endogenous kinase in a Ca²⁺/calmodulin-stimulated manner, including a M_r ca. 72,000 polypeptide found only in the transverse tubule.     

Ca2+-independent stimulation of cyclic GMP-dependent protein kinase by calmodulin

Calmodulin purified from bovine brain markedly stimulated cyclic GMP-dependent protein kinase from pig lung in the presence of cyclic GMP. This stimulation by calmodulin did not require Ca²⁺ and was dose-dependent up to optimal amounts, but the extent of stimulation decreased at concentrations over the optimal condition. The concentrations of cyclic GMP and cyclic AMP producing half-maximal stimulation were 4.5 × 10^?8 M and 5.0 × 10^?6 M respectively, under optimal conditions. Calmodulin increased maximum velocity without altering the Km for ATP. These effects of calmodulin on cyclic GMP-dependent protein kinase were similar to those of the stimulatory modulator described by Kuo and Kuo (J. Biol. Chem. $\text{251}$, 4283–4286, 1976). Ouf findings indicate that calmodulin regulates enzyme activity both Ca²⁺-dependently and independently.     

A multifunctional cyclic nucleotide- and Ca2+-independent protein kinase from rabbit skeletal muscle

A cyclic nucleotide- and Ca²⁺-independent protein kinase, initially identified as a glycogen synthase kinase (Itarte, E. and Huang, K.-P. (1979) J. Biol. Chem. $\text{254}$, 4052–4057), was also found to phosphorylate phosphorylase kinase and troponin from skeletal muscle as well as myosin light chain and myosin light chain kinase from both smooth and skeletal muscles. With the exception of myosin light chain from skeletal muscle, all the above-mentioned proteins are also substrates for the multifunctional cAMP-dependent protein kinase. The results suggest that this cyclic nucleotide- and Ca²⁺-independent protein kinase, like cAMP-dependent protein kinase, may have multiple cellular functions.     

(Ca2+ + Mg2+)-ATPase in enriched sarcolemma from dog heart

An enriched fraction of plasma membranes was prepared from canine ventricle by a process which involved thorough disruption of membranes by vigorous homogenization in dilute suspension, sedimentation of contractile proteins and mitochondria at $\text{3000 × g}$ followed by sedimentation of a microsomal fraction at $\text{200 000 × g}$. The microsomal suspension was then fractionated on a discontinuous sucrose gradient. Particles migrating in the density range 1.0591–1.1083 were characterized by ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity and [³H]ouabain binding as being enriched in sarcolemma and were comprised of nonaggregated vesicles of diameter approx. 0.1 μm. These fractions contained $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ which appeared endogenous to the sarcolemma. The enzyme was solubilized using Triton X-100 and 1 M KCl and partially purified. Optimal Ca²⁺ concentration for enzyme activity was 5–10 μM. Both Na⁺ and K⁺ stimulated enzyme activity. It is suggested that the enzyme may be involved in the outward pumping of Ca²⁺ from the cardiac cell.     

Purification of a water-soluble Mg2+-ATPase from human erythrocyte membranes

A water-soluble Mg²⁺-ATPase previously reported (White, M.D. and Ralston, G.B. (1976) Biochim. Biophys. Acta 436, 567–576) has been purified from human erythrocyte membranes. The purified enzyme has a molecular weight of 575 000; the apparent minimum molecular weight was 100 000, corresponding to a soluble protein of the component 3 region. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for ATP was 1 mM and apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg²⁺ was 3.6 mM. By means of histochemical activity staining in acrylamide gels it was shown that the purified ATPase preparation could be inhibited by Cd²⁺ and Zn²⁺ salts, $\text{p-}\text{chloromercuribenzoate}$ and $\text{N-}\text{ethylmaleimide}$, known inhibitors of membrane endocytosis.     

Protein substrate specificity of a calmodulin-dependent protein kinase isolated from bovine heart

The protein substrate specificity of a calmodulin-dependent protein kinase activity from the cytosolic fraction of bovine heart was examined. Prior to the experiments, the kinase activity was purified more than 50-fold with a recovery of greater than 10% of the homogenate activity. Two endogenous protein substrates of molecular weight 57,000 and 73,000 were phosphorylated in these kinase preparations. The kinase preparation was also able to phosphorylate exogenous synapsin, phospholamban, glycogen synthase, MAP-2, myelin basic proteins and κ-casein, but not tubulin, pyruvate kinase, the regulatory subunit of cAMP protein kinase II, myosin light chain or phosphorylase b. High levels of calmodulin were required for activation of the kinase activity toward the 57,000 and 73,000 molecular weight endogenous substrates (K_0.5 = 93 +/- 5 nM), glycogen synthase (K_0.5 = 127 +/- 10 nM), and κ-casein (K_0.5 = 321 +/- 107 nM). The kinase possessed a high affinity for glycogen synthase (half maximal activity at 0.9 +/- 0.4 μM) but a low affinity for κ-casein (21 +/- 2 μM). Sucrose density gradient centrifugation separated the calmodulin-dependent protein kinase activity into two fractions with apparent molecular weights of approximately 900,000 and 100,000. Both fractions phosphorylated the endogenous 57,000 molecular weight substrate and glycogen synthase similarly. These results indicate that cardiac calmodulin-dependent protein kinase previously observed to phosphorylate endogenous protein substrate possesses a wide range of substrate specificity.     

Characterization of a β-actinin-like protein in purified non-muscle cell membranes. Its activity on (Na+ + K+)-ATPase

Treatment by EDTA of purified plasma membranes from MF₂S cells (a variant of the murine plasmacytoma MOPC 173) solubilized proteins and increased by a 1000-fold the sensitivity of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ to ouabain. When added back with Ca²⁺ to treated plasma membranes, these EDTA-solubilized proteins restored the initial sensitivity of the enzyme to its inhibitor. We report the purification of a protein of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 32 000, isolated from the EDTA-treated membrane supernatant. This protein was purified by a one-step procedure involving a preparative polyacrylamide gel electrophoresis without detergent. In the presence of Ca²⁺ it was able to restore the original sensitivity to ouabain of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from EDTA-treated membrane. This protein was shown to be similar to the β-actinin described by Maruyama by the following criteria: (1) molecular weight and amino acid composition; (2) cross-reactivity with their respective antisera; (3) in the presence of Ca²⁺ the same quantitative biological activity on ouabain sensitivity of the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. A possible interaction between β-actinin, calmodulin and membrane-bound $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ is discussed.     

Regulation of calcium accumulation and efflux from platelet vesicles: Possible role for cyclic-AMP-dependent phosphorylation and calmodulin

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ activity of about 10 nmol (min·mg)^?1 and an ATP-dependent Ca²⁺ uptake of about 10 nmol (min·mg)^?1. When incubated in the presence of Mg[γ-³²P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [³²P]phosphate-labeled Ca²⁺ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca²⁺ or Ca²⁺ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of ¹²⁵I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol ($\text{M}{}_{\mathrm{r}}\text{= 94 000, 87 000, 60 000 and 43 000}$). Some minor calmodulin-binding proteins were enriched in the membrane fractions ($\text{M}{}_{\mathrm{r}}\text{= 69 000, 57 000, 39 000 and 37 000}$). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca²⁺ uptake is essentially unaltered, while the Ca²⁺ capacity is diminished as a consequence of a doubling in the rate of Ca²⁺ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca²⁺ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 μM calmodulin result in increased levels of vesicle phosphorylation.     

Calcium stimualtion of protein kinase C in the absence of added phospholipids

The activity of protein kinase C as isolated and described by Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. $\text{252}$, 7610–7616, can be markedly stimulated by Ca²⁺ in the presence of 4 mM Mg²⁺. This Ca²⁺ dependency does not require the presence of phospholipids or exogenous calmodulin. The increase in activity in the presence of Ca²⁺ is blocked by fluphenazine in the presence of 30 mM 2-mercaptoethanol. These results suggest that a calmodulin-like moiety may be a subunit of prokinase C.     

Phosphorylation of synaptosomal cytoplasmic proteins: Inhibition of calcium-activated,phospholipid-dependent protein kinase (protein kinase c) by bay k 8644

The phosphorylation of specific substrates of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was examined in striatal synaptosomal cytoplasm. The phosphoprotein substrata were termed group C phosphoprotems and were divided into two subgroups: group C₁ phosphoproteins (P83, P45A, P21 and P18) were found in both cytoplasm and synaptosomal membranes and, although stimulated by phosphatidylserine, only required exogamous calcium for their labeling; group C₂ phosphoproteins (P120, P96, P21.5, P18.5 and P16) were found predominantly in the cytoplasm and were absolutely dependent upon exogenous calcium and phosphatidylserme for their labeling. Several criteria were used to identify these proteins as specific protein kinase C substrates: (a) their phosphorylation was stimulated to a greater extent by Ca²⁺ /phosphatidylserine/diolein than by Ca²⁺ alone or Cal²⁺ /calmodulin (group C₁) or was completely dependent upon Ca²⁺ /phosphatdylserine/diolein (group C₂); (b) supermaximal concentrations of the cAMP-dependent protein kinase inhibitor were without effect; (c) their phosphorylation was stimulated by oleic acid, which selectively activates protein kinase C in the absence of Ca²⁺; (d) NaCl, which inhibited cAMP- and Ca²⁺/calmodulindependent phosphorylation, slightly increased phosphorylation of group C₁ and slightly decreased phosphorylation of group C₂ phosphoproteins. Maximal phosphorylation of P96 and other group C phosphoproteins occurred within 60 s and was followed by a slow decay rate while substrata of calmodulin-dependent protein kinase were maximally labeled within 20–30 s and rapidly dephosphorylated. The phosphorylation of all group C phosphoproteins was inhibited by the calcium channel agomst BAY K 8644, however, group C₂ phosphoproteins were considerably more sensitive. The IC₅₀ for inhibition of P96 labeling was 19 μM. but for P83 was 190 μM. Group B phosphoproteins were also slightly inhibited, and the IC₅₀ for P63 was 290 μM. No inhibitory effects of another dihydropyridine, nifedipine, or of verapamil were detected in this concentration range. BAY K 8644 did not displace [³H]phorbol-12,13-dibutyrate binding, nor was the inhibition decreased by increasing phosphatidylserine concentrations. BAY K 8644 had no effect on the rate of dephosphorylation of any phosphoprotein, indicating that it is unlikely to inhibit a protein phosphatase. BAY K 8644 may, therefore, prove to be a valuable tool for discriminating protein kinase C activity from the activity of other protein kinases. We conclude that BAY K 8644 interacts either with a specific subgroup of protein kinase C substrata or with one of two putative forms of protein kinase C.     

Effects of prostaglandin PGE2 on the synthesis of placental proteins and human placental lactogen (HPL)

The biosynthesis of placental proteins and placental lactogen (HPL) was studied $\text{in vitro}$ in 10–12 week, 16–18 week and term human placenta in the presence and absence of PGE_2α. The highest ¹⁴C-leucine incorporation was detected in 10 to 12 weeks old placentas. Addition of PGE_2α to the induction medium depressed the rate of incorporation of ¹⁴C-leucine into placental proteins on a dose dependent manner. Placentas most sensitive to this action of PGE_2α were those obtained at 18 weeks gestation followed by placentas at term. $\text{In vivo}$ application of PGE_2α for tharapeutic induction of abortions resulted in the marked inhibition of placental protein synthesis $\text{in vitro}$.     

Purification and characterization of a calcium-dependent protein kinase from beetroot plasma membranes

Several calcium-dependent protein kinases (CDPKs) are located in plant plasma membranes where they phosphorylate enzymes and transporters, like the H⁺-ATPase and water channels, thereby regulating their activities. In order to determine which kinases phosphorylate the H⁺-ATPase, a calcium-dependent kinase was purified from beetroot (Beta vulgaris L.) plasma membranes by anion-exchange chromatography, centrifugation in glycerol gradients and hydrophobic interaction chromatography. The kinetic parameters of this kinase were determined (V _max: 3.5 μmol mg⁻¹ min⁻¹, K _m for ATP: 67 μM, K _m for syntide 2: 15 μM). The kinase showed an optimum pH of 6.8 and a marked dependence on low-micromolar Ca²⁺ concentrations (K _d : 0.77 μM). During the purification procedure, a 63-kDa protein with an isoelectric point of 4.7 was enriched. However, this protein was shown not to be a kinase by mass spectrometry. Kinase activity gels showed that a 50-kDa protein could be responsible for most of the activity in purified kinase preparations. This protein was confirmed to be a CDPK by mass spectrometry, possibly the red beet ortholog of rice CDPK2 and Arabidopsis thaliana CPK9, both found associated with membranes. This kinase was able to phosphorylate purified H⁺-ATPase in a Ca²⁺-dependent manner.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

The stereochemical course of thiophosphoryl group transfer catalyzed by adenosine kinase

Adenosine kinase was partially purified form beef liver and used to catalyze the conversion of (γR)ATPγS,γ¹⁸O and adenosine to ADP and AMPαS,α¹⁸O. The configuration at phosphorus in AMPαS,α¹⁸O was established by subjecting it to stereospecific phosphorylation to (αS)ATPαS,α¹⁸O and showing that only the nonbridging oxygen bonded to the α-P was enriched with ¹⁸O. The configuration at α-P in AMPαS,α¹⁸O was therefore S, and the transfer of the [¹⁸O]thiophosphoryl group occurred with $\text{inversion}$ of configuration.