Identification and characterization ofThermoplasma acidophilum 2-keto-3-deoxy-D-gluconate kinase: A new class of sugar kinases

The thermoacidophilic archaeonThermoplasma acidophilum has long been known to utilized-glucosevia the non-phosphorylated Entner-Doudoroff (nED) pathway. We now report the identification of a gene encoding 2-keto-3-deoxy-d-gluconate (KDG) kinase. The discovery of this gene implies the presence of a glycolysis pathway, other than the nED pathway. It was found that Ta0122 in theT. acidophilum genome corresponded to KDG kinase. This enzyme shares no similarity with known KDG kinases, and belongs to a novel class of sugar kinases. Of the five sugars tested only KDG was utilized as a substrate.     

A MOFRL family glycerate kinase from the thermophilic crenarchaeon, <Emphasis Type="Italic">Sulfolobus tokodaii</Emphasis>, with unique enzymatic properties

A glycerate kinase gene (ST2037) from the hyperthermophilic crenarchaeon Sulfolobus tokodaii was cloned and expressed in Escherichia coli. The purified homodimeric protein (45 kDa) specifically catalyzed the formation of 2-phosphoglycerate with d-glycerate as substrate. The thermostable enzyme displayed maximum activity (over 20 min) at 90°C and pH 4.5. The maximal activity was in the presence of Co²⁺. The MOFRL family glycerate kinase used AMP as phosphate donor with maximal activity towards GTP. These characteristics of the enzyme suggested its potential in the catalytic production of 2-phosphoglycerate.     

A unique highly thermostable 2-phosphoglycerate forming glycerate kinase from the hyperthermophilic archaeon <Emphasis Type="Italic">Pyrococcus horikoshii</Emphasis>: gene cloning,expression and characterization

A glycerate kinase (GK) gene (PH0495) from the hyperthermophilic archaeon Pyrococcus horikoshii, was cloned and expressed in Escherichia coli. The recombinant protein was purified to homogeneity by affinity chromatography and ion exchange chromatography. The enzyme was likely a homodimer based on SDS-PAGE (47 kDa) and gel filtration chromatography (100 kDa) analysis. A radioisotope-labeling examination method was initially used for the enzymatic activity detection, and the enzyme (GK_ph) was found to catalyze the formation of 2-phosphoglycerate using d-glycerate as the substrate. The enzyme exhibited unique phosphoryl donor specificity with maximal activity towards pyrophosphate. The temperature and pH optima of the enzyme were 45°C and 7.0, respectively, and about half of the maximal activity remained at 100°C. The enzyme was highly thermostable with almost no loss of activity at 90°C for 12 h. Based on sequence alignment and structural comparison it was assigned to group I of the trichotomy of GKs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A thermostable phosphotriesterase from the archaeon Sulfolobus solfataricus: cloning, overexpression and properties

A new gene from the hyperthermophilic archaeon Sulfolobus solfataricus MT4, coding for a putative protein reported to show sequence identity with the phosphotriesterase-related protein family (PHP), was cloned by means of the polymerase chain reaction from the S. solfataricus genomic DNA. In order to analyse the biochemical properties of the protein an overexpression system in Escherichia coli was established. The recombinant protein, expressed in soluble form at 5 mg/l of E. coli culture, was purified to homogeneity and characterized. In contrast with its mesophilic E. coli counterpart that was devoid of any tested activity, the S. solfataricus enzyme was demonstrated to have a low paraoxonase activity. This activity was dependent from metal cations with Co²⁺, Mg²⁺ and Ni²⁺ being the most effective and was thermophilic and thermostable. The enzyme was inactivated with EDTA and o-phenantroline. A reported inhibitor for Pseudomonas putida phosphotriesterase (PTE) had no effect on the S. solfataricus paraoxonase. The importance of a stable paraoxonase for detoxification of chemical warfare agents and agricultural pesticides will be discussed.     

Purification and characterization of D-glycerate-3-kinase from maize leaves

Glycerate kinase (GK; EC 2.7.1.31) from maize (Zea mays L.) leaves was purified by a sequence of ammonium-sulfate precipitations and chromatography on diethylaminoethyl-cellulose, hydroxyapatite, Sephadex G-75SF and dye ligand (Green A) columns. The purest preparation was almost 1300-fold enriched and had a specific activity of 68 mol · min^-1 · (mg protein) ^-1. The enzyme was a monomer of a relative molecular mass (M_r) of 44 kDa (kdalton) as determined by gel filtration, electrophoresis in dissociating conditions and by immunoblots. The enzyme was only weakly recognized by polyclonal antibodies against purified spinach GK, indicating substantial differences in molecular structure of the two proteins. Highly reducing conditions stabilized GK activity and were required for activation of crude leaf enzyme. The enzyme had a broad pH optimum of 6.8–8.5, and formed 3-phosphoglycerate and ADP as reaction products. Apparent K _ms for D-glycerate and Mg-ATP were 0.11 and 0.25 mM, respectively. The enzyme was strongly affected by a number of phosphoesters, especially by 3-phosphoglycerate (K _i= 0.36 mM), fructose bisphosphates and nucleoside bisphosphates. Inhibition by 3-phosphoglycerate was competitive to Mg-ATP and noncompetitive to D-glycerate. Pyruvate was found noncompetitive to D-glycerate (K _is=4 mM). The ratio of stromal concentration of Mg-ATP to phosphoesters, particularly to 3-phosphoglycerate, may be of importance in the regulation of GK during C₄-photosynthesis.Abbreviations DEAE diethylaminoethyl - kDa kdalton - GAP-DH glyceraldehyde phosphate dehydrogenase - GK glycerate kinase - LDH lactate dehydrogenase - 2-ME 2-mercaptoethanol - M_r relative molecular mass - PEP phosphoenolpyruvate - PGA(PK) phosphoglycerate (phosphokinase) - PK pyruvate kinase - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis     

Regulation of sedoheptulose-1,7-bisphosphatase by sedoheptulose-7-phosphate and glycerate,and of fructose-1,6-bisphosphatase by glycerate in spinach chloroplasts

Using partially purified sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea L.) chloroplasts the effects of metabolites on the dithiothreitoland Mg²⁺-activated enzyme were investigated. A screening of most of the intermediates of the Calvin cycle and the photorespiratory pathway showed that physiological concentrations of sedoheptulose-7-phosphate and glycerate specifically inhibited the enzyme by decreasing its maximal velocity. An inhibition by ribulose-1,5-bisphosphate was also found. The inhibitory effect of sedoheptulose-7-phosphate on the enzyme is discussed in terms of allowing a control of sedoheptulose-1,7-bisphosphate hydrolysis by the demand of the product of this reaction. Subsequent studies with partially purified fructose-1,6-bisphosphatase from spinach chloroplasts showed that glycerate also inhibited this enzyme. With isolated chloroplasts, glycerate was found to inhibit CO₂ fixation by blocking the stromal fructose-1,6-bisphosphatase. It is therefore possible that the inhibition of the two phosphatases by glycerate is an important regulatory factor for adjusting the activity of the Calvin cycle to the ATP supply by the light reaction.Abbreviations DTT dithiothreitol - FBPase fructose-1,6-bisphosphatase - Fru-1,6-P₂ fructose-1,6-bisphosphate - Fru-6-P fructose-6-phosphate - 3-PGA 3-phosphoglycerate - Ru-1,5-P₂ ribulose-1,5-bisphosphate - Ru-5-P ribulose-5-phosphate - SBPase sedoheptulose-1,7-bisphosphatase - Sed-1,7-P₂ sedoheptulose-1,7-bisphosphate - Sed-7-P sedoheptulose-7-phosphate This work was supported by the Deutsche Forschungsgemein-schaft.     

Solubilization, partial purification, and reconstitution of the glycolate/glycerate transporter from chloroplast inner envelope membranes

The glycolate/glycerate transporter of spinach (Spinacia oleracea L.) chloroplast inner envelope membranes was solubilized by treatment of the membranes with sodium cholate. Mixtures of the cholate extracts and soy asolectin were subjected to gel filtration to remove the detergent. The reconstituted vesicles were frozen, thawed, and sonicated in a buffer that contained 10 millimolar d-glycerate and, usually, [³H]sucrose as an internal space indicator. The dilution of the vesicles into a medium that contained 0.4 millimolar [¹⁴C]d-glycerate resulted in a rapid accumulation of labeled glycerate, followed by a much slower loss of [¹⁴C]d-glycerate from the vesicles. This behavior is characteristic of counterflow. The accumulation of [¹⁴C]d-glycerate was strongly inhibited by HgCl₂, which blocks glycolate/glycerate transport in intact chloroplasts. In the absence of proton ionophores, the extent of [¹⁴C]glycolate accumulation under similar conditions was much greater than that of [¹⁴C]d-glycerate. External glycolate inhibited d-glycerate counterflow and external d-glycerate inhibited glycolate counterflow. The external pH dependence of the efflux of [¹⁴C]d-glycerate accumulated in vesicles by counterflow and its inhibition by external l-mandelate are characteristics displayed by glycolate transport in intact chloroplasts. Partial purification of the transporter was achieved by glycerol gradient centrifugation. The solubilized glycolate and glycerate counterflow activities, assayed by reconstitution into vesicles, were found to sediment similarly.     

Cloning, expression and characterization of a thermostable exo-β-D-glucosaminidase from the hyperthermophilic archaeon Pyrococcus horikoshii

An exo-β-d-glucosaminidase gene (PH0511) was cloned from the hyperthermophilic archaeon, Pyrococcus horikoshii, and expressed in Escherichia coli. The purified protein showed a strong exo-β-d-glucosaminidase activity by TLC analysis. DTT (50 mM) had little effect on its homodimeric structure during SDS-PAGE. The enzyme was optimally active at 90°C (over 20 min) and pH 6. It had a half-life of 9 h at 90°C and is the most thermostable glucosaminidase described up to now. The activity was not inhibited by ethanol, 2-propanol, DMSO, PEG-400, denaturing agents SDS (5%, w/v), urea, guanidine hydrochloride (5 M) and Mg²⁺, Mn²⁺, Co²⁺, Ca²⁺, Sr²⁺, Ni²⁺ (at up to 10 mM).     

Kinetic and regulatory properties of pyruvate kinase isozymes from flight muscle and fat body of the cockroach,Periplaneta americana

Summary Pyruvate kinases from flight muscle and fat body of the cockroach,Periplaneta americana, were purified to homogeneity. The two tissues contained different forms of the enzyme which were separable by starch gel electrophoresis and isoelectric focusing (pI=5.75 for flight muscle and 6.15 for fat body). Both enzymes had molecular weights of 235,000±20,000.Flight muscle pyruvate kinase displayed Michaelis-Menten kinetics with respect to both ADP and P-enolpyruvate withK _m values of 0.27 and 0.04 mM, respectively.K _m for Mg²⁺ was 0.60 mM andK _a for K⁺ was 15 mM. The enzyme was weakly inhibitied by four compounds, ATP, arginine-P,l-alanine and citrate with apparentK _i values of 3.5, 15, 20 and 24 mM, respectively. Competitive inhibition by 3 mM ATP or 10 mM arginine-P raised theK _m for P-enolpyruvate to 0.067 or 0.057 mM. Fructose-1,6-P₂ did not activate the enzyme but reversed inhibitions by ATP and arginine-P.Fat body pyruvate kinase showed sigmoidal kinetics with respect to P-enolpyruvate with S_0.5=0.32 mM andn _H=1.43.K _m values for ADP and Mg²⁺ were 0.30 and 0.80 mM, respectively with aK _a for K⁺ of 10 mM. ATP andl-alanine were inhibitors of the enzyme; 2 mM ATP raised S_0.5 for P-enolpyruvate to 0.48 mM while 3 mMl-alanine increased S_0.5 to 0.84 mM. Neither citrate nor arginine-P inhibited the enzyme but citrate affected the enzyme by reversingl-alanine inhibition. Fat body pyruvate kinase was strongly activated by fructose-1,6-P₂ with an apparentK _a of 1.5 M. Fructose-1,6-P₂ at 0.1 mM reduced S_0.5 for P-enolpyruvate to 0.05 mM andn _H to 1.0.Flight muscle and fat body pyruvate kinases from the cockroach show properties analogous to those of the muscle and liver forms of mammalian pyruvate kinase. Fat body pyruvate kinase is suited for on-off function in a tissue with a gluconeogenic capacity. Strong allosteric control with a feed-forward activation by fructose-1,6-P₂ is key to coordinating enzyme function with glycolytic rate. The function of flight muscle pyruvate kinase in energy production during flight is aided by a lowK _m for P-enolpyruvate, weak inhibitor effects by high energy phosphates and deinhibition of these effects by fructose-1,6-P₂.     

Inhibition of ATPases Enzyme Activities on Brain Disturbing Normal Oestrous Cycle

Wistar rats treated with -methyl- DL-p -tyrosine methylester showed significant level of inhibition in the activity of Na⁺, K⁺-ATPase, Mg²⁺-ATPase and Ca²⁺-ATPase enzymes in different regions of the brain. The enzyme activity was assayed in cerebral hemispheres, hypothalamus, thalamus, hippocampus, amygdala and septum at proestrous (12 h), estrous (25 h), metestrous (38 h) and diestrous periods (92 h) of the rat. The Na⁺, K⁺-ATPase activity was significantly inhibited in most of the brain regions after treated with -methyl- DL-p -tyrosine methylester (MPT) and this indicated that MPT affected the active transport system and nerve impulse transmission. Mg²⁺-ATPase and Ca²⁺-ATPase was also significantly (P < 0.001) reduced in different regions of the brain. The results revealed that MPT affected active transport system and nerve impulse transmission by inhibiting Na⁺, K⁺-ATPase and Ca²⁺-ATPase. It has induced energy crisis by inhibiting Mg²⁺-ATPase and all these cumulative effects of MPT have adversely affected the female Wistar rats. These effects have been manifested in the form of aberrations in the behavior of MPT treated female rats, which have shown their inability to perform their normal sexual activity.     

Novel thermoactive glucoamylases from the thermoacidophilic Archaea Thermoplasma acidophilum,Picrophilus torridus and Picrophilus oshimae

The thermoacidophilic Archaea Thermoplasma acidophilum (optimal growth at 60 °C and pH 1–2), Picrophilus torridus and Picrophilus oshimae (optimal growth at 60 °C and pH 0.7) were able to utilize starch as sole carbon source. During growth these microorganisms secreted heat and acid-stable glucoamylases into the culture fluid. Applying SDS gel electrophoresis activity bands were detected with appearent molecular mass (Mw) of 141.0, 95.0 kDa for T. acidophilum, 133.0, 90.0 kDa for P. torridus and 140.0, 85.0 kDa for P. oshimae. The purified enzymes were incubated with various polymeric substrates such as starch, pullulan, panose and isomaltose. The product pattern, analyzed by HPLC, showed that in all cases glucose was formed as the sole product of hydrolysis. The purified glucoamylases were optimally active at pH 2.0 and 90 °C and have an isoelectric points (pI) between 4.5 and 4.8. Enzymatic activity was detected even at pH 1.0 and 100 °C. The glucoamylases were thermostable at elevated temperature with a half-life of 24 h at 90 °C for both P. torridus and T. acidophilum, and 20 h at 90 °C for P. oshimae. The enzyme system of T. acidophilum has a lower K _m value for soluble starch (1.06 mg/ml) than the enzymes from P. oshimae and P. torridus (4.35 mg/ml and 2.5 mg/ml), respectively. Enzyme activity was not affected by Na⁺, Mg⁺⁺, Ca⁺⁺, Ni⁺⁺, Zn⁺⁺, Fe⁺⁺, EDTA and DTT. This revised version was published online in August 2006 with corrections to the Cover Date.     

Thiol-dependent regulation of glycerate metabolism in leaf extracts : the role of glycerate kinase in c(4) plants

We have recently reported that the activity of maize leaf glycerate kinase [EC 2.7.1.31] is regulated in vivo by the light/dark transition, possibly involving the ferredoxin/thioredoxin mechanism, and that the stimulating effect of light can be mimicked in vitro by incubation of crude leaf extract with reducing compounds (LA Kleczkowski, DD Randall 1985 Plant Physiol 79: 274-277). In the present study it was found that the time course of thiol activation of the enzyme was substantially dependent on the presence of some low molecular weight inhibitor(s) of activation found both in leaf extracts and mesophyll chloroplasts. Activity of glycerate kinase from maize as well as wheat leaves increased upon greening of etiolated plants and was correlated with the development of photosynthetic apparatus in these species. The maize enzyme was strongly activated by thiols at all stages of development from etiolated to green seedlings. Thiol activation of glycerate kinase was observed for a number of C₄ plants, notably of the nicotinamide adenine dinucleotide phosphate-malic enzyme type, with the strongest effect found for the enzyme from leaf extracts of maize and sorghum (10- and 8-fold activation, respectively). Among the C₃ species tested, only the enzyme from soybean leaves was affected under the same conditions (1.6-fold activation). This finding was reflected by an apparent lack of cross-reactivity between the enzyme from maize leaves and antibodies raised against purified spinach leaf glycerate kinase. We suggest that, in addition to its role as a final step of photorespiration in leaves, glycerate kinase from C₄ species may serve as a part of the facilitative diffusion system for the intercellular transport of 3-phosphoglycerate. Simultaneous operation of both the passive and the facilitative diffusion mechanisms of 3-phosphoglycerate transport in C₄ plants is postulated.     

The pathway ofD-cycloserine biosynthesis inStreptomyces garyphalus

Incubation experiments using washed cells and toluene treated cells ofStreptomyces garyphalus showed that O-acetyl-L-serine and hydroxyurea are intermediates in the biosynthesis ofD-cycloserine. The formation of [¹⁴C]O-ureidoserine from O-acetyl-L-serine and hydroxyurea was demonstrated by incubating an enzyme solution with¹⁴C-labelled substrates. Desalted cell-free extract catalyzed the conversion of O-ureido-D-serine toD-cycloserine in a reaction requiring ATP and Mg²⁺. The results suggested the following pathway forD-cycloserine biosynthesis.     

Cloning and overexpression in Escherichia coli of the gene encoding citrate synthase from the hyperthermophilic Archaeon Sulfolobus solfataricus

The citrate synthase (CS) gene from the hyperthermophilic Archaeon Sulfolobus solfataricus has been cloned and sequenced. The gene encodes a polypeptide of 378 amino acids with a calculated polypeptide molecular mass of 42 679. High-level expression was achieved in Escherichia coli and the recombinant citrate synthase was purified to homogeneity using a heat step and dye-ligand affinity chromatography. This procedure yielded approximately 26 mg of pure CS per liter of culture, with a specific activity of 41 U/mg. The enzyme exhibited a half-life of 8 min at 95°C. A homology-modelled structure of the S. solfataricus CS has been generated using the crystal structure of the enzyme from the thermoacidophilic Archaeon Thermoplasma acidophilum with which it displays 58% sequence identity. The modelled structure is discussed with respect to the thermostability properties of the enzyme. Received: August 10, 1997 / Accepted: October 23, 1997     

Purification and characterization of naringinase from a newly isolated strain of <Emphasis Type="Italic">Aspergillus niger</Emphasis> 1344 for the transformation of flavonoids

Summary An extracellular naringinase (an enzyme complex consisting of α-L-rhamnosidase and β-D-glucosidase activity, EC 3.2.1.40) that hydrolyses naringin (a trihydroxy flavonoid) for the production of rhamnose and glucose was purified from the culture filtrate of Aspergillus niger 1344. The enzyme was purified 38-fold by ammonium sulphate precipitation, ion exchange and gel filtration chromatography with an overall recovery of 19% with a specific activity of 867 units per mg of protein. The molecular mass of the purified enzyme was estimated to be about 168 kDa by gel filtration chromatography on a Sephadex G-200 column and the molecular mass of the subunits was estimated to be 85 kDa by sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 4.0 and temperature of 50 °C, respectively. The naringinase was stable at 37 °C for 72 h, whereas at 40 °C the enzyme showed 50% inactivation after 96 h of incubation. Hg²⁺, SDS, p-chloromercuribenzoate, Cu²⁺ and Mn²⁺ completely inhibited the enzyme activity at a concentration of 2.5–10 mM, whereas, Ca²⁺, Co²⁺ and Mg²⁺ showed very little inactivation even at high concentrations (10–100 mM). The enzyme activity was strongly inhibited by rhamnose, the end product of naringin hydrolysis. The enzyme activity was accelerated by Mg²⁺ and remained stable for one year after storage at −20 °C. The purified enzyme preparation successfully hydrolysed naringin and rutin, but not hesperidin.     

Light and thiol activation of maize leaf glycerate kinase : the stimulating effect of reduced thioredoxins and ATP

Glycerate kinase (EC 2.7.1.31) from maize (Zea mays) leaves was shown to be regulated by light/dark transition. The enzyme more than doubled in activity after either the leaves or isolated mesophyll chloroplasts were illuminated with white light for 10 minutes. Rate of inactivation in the dark was faster in leaves than in the isolated chloroplast fraction. The stimulating effect of light could be mimicked in crude preparations by addition of 10 or 50 millimolar dithiothreitol or 100 millimolar 2-mercaptoethanol. The thiol treatment resulted in 8- to 10-fold activation of glycerate kinase, with the highest rates in the range of 27 to 30 micromoles per mg chlorophyll per hour. Activation was not accompanied by any changes in the apparent M_r value of glycerate kinase as determined by gel filtration (M_r = 47,000). In contrast to maize glycerate kinase, the enzyme from spinach was not affected by either light or thiol exposure.

Partially purified maize glycerate kinase was activated up to 3-fold upon incubation with a mixture of spinach thioredoxins m and f and 5 millimolar dithiothreitol. The thioredoxin and dithiothreitol-treated glycerate kinase could be further stimulated by addition of 2.5 millimolar ATP. The results suggest that glycerate kinase from maize leaves is capable of photoactivation by the ferredoxin/thioredoxin system. The synergistic effect of ATP and thioredoxins in activation of the enzyme supports the earlier expressed view that the ferredoxin/thioredoxin system functions jointly with effector metabolites in light-mediated regulation during photosynthesis.

     

Optimization of medium for extracellular nuclease formation from Rhizopus stolonifer

A strain of Rhizopus stolonifer produced a high activity of extracellular DNAase when grown on YPG (yeast extract peptone glucose) medium. The source of peptone had a marked effect on the enzyme yield and only one peptone (i.e. from Sarabhai M. Chemicals Ltd, India) supported enzyme production. Maximum enzyme activity (88 U/ml) was obtained after 4 days' growth under submerged conditions in YPG medium containing 100 M Mn²⁺, Co²⁺ or Mg²⁺, and glucose as the sole carbon source. The unpurified enzyme was optimally active at pH 7.5 and 45°C. It had a higher activity with sonicated and heat-denatured DNA than native DNA.     

Relationship between Calcium and Pyruvate Kinase

Tobacco plants (Nicotiana tabacum L. cv. Sevilla) were grown under controlled conditions. The leaf content of Ca²⁺, Mg²⁺ and K⁺, and the activity of the pyruvate kinase were analyzed. The increased application of Ca²⁺ diminished the content of K⁺ and Mg²⁺ in leaves, and decreased the activity of pyruvate kinase. Taking into account these results, we suggest the pyruvate kinase activity as an bioindicator of the contents of the Ca²⁺, Mg²⁺ and K⁺.     

Structural analysis of the plasmid pTA1 isolated from the thermoacidophilic archaeon Thermoplasma acidophilum

Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at pH1.8 and 56°C and has no cell wall. Plasmid pTA1 was found in some strains of the species. We sequenced plasmid pTA1 and analyzed the open reading frames (ORFs). pTA1 was found to be a circular DNA molecule of 15,723 bp. Eighteen ORFs were found; none of the gene products except ORF1 had sequence similarity to known proteins. ORF1 showed similarity to Cdc6, which is involved in genome-replication initiation in Eukarya and Archaea. T. acidophilum has two Cdc6 homologues in the genome. The homologue found in pTA1 is most similar to Tvo3, one of the three Cdc6 homologues found in the genome of Thermoplasma volcanium, among all of the Cdc6 family proteins. The phylogenetic analysis suggested that plasmid pTA1 is possibly originated from the chromosomal DNA of Thermoplasma.     

Effect of Mg2+ concentrations on phosphorylation/activation of phosphorylase b kinase by cAMP/Ca2+-independent, autophosphorylation-dependent protein kinase

In a previous report (Yu and Yang,Biochem. Biophys. Res. Commun. 207, 140–147 (1995)], phosphorylase b kinase from rabbit skeletal muscle was found to be phosphorylated and activated by a cyclic nucleotide- and Ca²⁺-independent protein kinase previously identified as an autophosphorylation-dependent multifunctional protein kinase (autokinase) from brain and liver (Yanget al, J. Biol. Chem. 262, 7034–7040, 9421–9427 (1987)]. In this report, the effect of Mg²⁺ ion concentration on the auto-kinase-catalyzed activation of phosphorylase b kinase is investigated. The levels of phosphorylation and activation of phosphorylase b kinase catalyzed by auto-kinase are found to be dependent on the concentration of Mg²⁺ ion used. Phosphorylation of phosphorylase b kinase at high Mg²⁺ ion (>9 mM) is 2–3 times higher than that observed at low Mg²⁺ ion (1 mM) and this results in a further 2- to 3-fold activation of the enzyme activity at high Mg²⁺ ion. Analysis of the phosphorylation stoichiometry of and subunits of phosphorylase b kinase at different Mg²⁺ ion concentrations further reveals that the phosphorylation level of the subunit remains almost unchanged, whereas the phosphorylation level of the subunit increases dramatically and correlates with the increased enzyme activity. In similarity with the subunit, phosphorylations of myelin basic protein and histone 2A by auto-kinase are also unaffected by Mg²⁺ ion. Taken together, the results provide initial evidence that Mg²⁺ ion may specifically render thea subunit a better substrate for auto-kinase to cause further phosphorylation/activation of phosphorylase b kinase, representing a new mode of control mechanism for the regulation of auto-kinase involved in the phosphorylation and concurrent activation of phosphorylase b kinase.