Creatine kinase regulation by reversible phosphorylation in frog muscle

Creatine kinase (CK) was analyzed from skeletal muscle of wood frogs, Rana sylvatica, a species that survives natural whole body freezing during the winter months. Muscle CK activity increased by 35% and apparent K_m creatine decreased by 29% when frogs froze. Immunoblotting analysis showed that this activity increase was not due to a change in total CK protein. Frog muscle CK was regulated by reversible protein phosphorylation; in vitro incubations with ³²P-ATP under conditions that facilitated the actions of various protein kinases (PKA, PKG, PKC, CaMK or AMPK) resulted in immunoprecipitation of ³²P-labeled CK. Furthermore, incubations that stimulated CaMK or AMPK altered CK kinetics. Incubation under conditions that facilitated protein phosphatases (PP2B or PP2C) reversed these effects. Phosphorylation of CK increased activity, whereas dephosphorylation decreased activity. Ion-exchange chromatography revealed that two forms of CK with different phosphorylation states were present in muscle; low versus high phosphate forms dominated in muscle of control versus frozen frogs, respectively. However, CK from control versus frozen frogs showed no differences in susceptibility to urea denaturation or sensitivity to limited proteolysis by thermolysin. The increased activity, increased substrate affinity and altered phosphorylation state of CK in skeletal muscle from frozen frogs argues for altered regulation of CK under energy stress in ischemic frozen muscle.     

Vertebrate freezing survival: Regulation of the multicatalytic proteinase complex and controls on protein degradation

The wood frog, Rana sylvatica, survives weeks of whole body freezing during winter hibernation, expressing numerous metabolic adaptations that deal not only with freezing but with its consequences including organ ischemia and cellular dehydration. The present study analyzes the 20s multicatalytic proteinase (MCP) complex from skeletal muscle to determine how protein degradation is managed in the ischemic frozen state. MCP was partially purified and assayed fluorometrically using three AMC-labeled substrates to compare multiple states: control (5 degrees C acclimated), 24 h frozen at -2.5 degrees C, 4 or 8 h thawed at 5 degrees C, 8 h anoxia, and 40% dehydration. MCP from frozen frogs showed significantly different K(m) and V(max) values compared with controls; e.g., K(m) Z-LLE-AMC increased by 45% during freezing and 52% under anoxia whereas V(max) decreased by 40%. After thawing, K(m) was restored and V(max) rose by 2.2-fold. Incubations promoting protein kinase or phosphatase action on MCP showed that phosphatase treatment strongly increased V(max) implicating reversible phosphorylation in MCP regulation during freeze-thaw. Western blotting showed a 36% decrease in MCP protein in muscle from frozen frogs. The 20s MCP preferentially degrades oxidatively-damaged proteins and evidence of impaired function during freezing came from a 1.4-fold increase in protein carbonyl content in muscle and liver during freezing. Ubiquitin and ubiquitin conjugate levels were unchanged in muscle but changed markedly in liver during freeze-thaw.     

Phosphorylation of recombinant human ATP:citrate lyase by cAMP-dependent protein kinase abolishes homotropic allosteric regulation of the enzyme by citrate and increases the enzyme activity. Allosteric activation of ATP:citrate lyase by phosphorylated sugars

Recombinantly expressed human ATP:citrate lyase was purified from E. coli, and its kinetic behavior was characterized before and after phosphorylation. Cyclic AMP-dependent protein kinase catalyzed the incorporation of only 1 mol of phosphate per mole of enzyme homotetramer, and glycogen synthase kinase-3 incorporated an additional 2 mol of phosphate into the phosphorylated protein. Isoelectric focusing revealed that all of the phosphates were incorporated into only one of the four enzyme subunits. Phosphorylation resulted in a 6-fold increase in V(max) and the conversion of citrate dependence from sigmoidal, displaying negative cooperativity, to hyperbolic. The phosphorylated recombinant enzyme is more similar to the enzyme isolated from mammalian tissues than unphosphorylated enzyme with respect to the K(m) for citrate, CoA, and ATP, and the specific activity. Fructose 6-phosphate was found to be a potent activator (60-fold) of the unphosphorylated recombinant enzyme, with half-maximal activation at 0.16 mM, which results in a decrease in the apparent K(m) for citrate and ATP, as well as an increase in the V(max) of the reaction. Thus, human ATP:citrate lyase activity is regulated in vitro allosterically by phosphorylated sugars as well as covalently by phosphorylation.     

Discovery of a novel site regulating glucokinase activity following characterization of a new mutation causing hyperinsulinemic hypoglycemia in humans

Type 2 diabetes is a global problem, and current ineffective therapeutic strategies pave the way for novel treatments like small molecular activators targeting glucokinase (GCK). GCK activity is fundamental to beta cell and hepatocyte glucose metabolism, and heterozygous activating and inactivating GCK mutations cause hyperinsulinemic hypoglycemia (HH) and maturity onset diabetes of the young (MODY) respectively. Over 600 naturally occurring inactivating mutations have been reported, whereas only 13 activating mutations are documented to date. We report two novel GCK HH mutations (V389L and T103S) at residues where MODY mutations also occur (V389D and T103I). Using recombinant proteins with in vitro assays, we demonstrated that both HH mutants had a greater relative activity index than wild type (6.0 for V389L, 8.4 for T103S, and 1.0 for wild type). This was driven by an increased affinity for glucose (S(0.5), 3.3 ± 0.1 and 3.5 ± 0.1 mm, respectively) versus wild type (7.5 ± 0.1 mm). Correspondingly, the V389D and T103I MODY mutants had markedly reduced relative activity indexes (<0.1). T103I had an altered affinity for glucose (S(0.5), 24.9 ± 0.6 mm), whereas V389D also exhibited a reduced affinity for ATP and decreased catalysis rate (S(0.5), 78.6 ± 4.5 mm; ATP(K(m)), 1.5 ± 0.1 mm; K(cat), 10.3 ± 1.1s(-1)) compared with wild type (ATP(K(m)), 0.4 ± <0.1; K(cat), 62.9 ± 1.2). Both Thr-103 mutants showed reduced inhibition by the endogenous hepatic inhibitor glucokinase regulatory protein. Molecular modeling demonstrated that Thr-103 maps to the allosteric activator site, whereas Val-389 is located remotely to this position and all other previously reported activating mutations, highlighting α-helix 11 as a novel region regulating GCK activity. Our data suggest that pharmacological manipulation of GCK activity at locations distal from the allosteric activator site is possible.     

Mechanisms of gamma-glutamylcysteine ligase regulation

The principal objective of this study was to investigate the mechanisms regulating the activity of gamma-glutamylcysteine ligase (GCL; EC 6.3.2.2), the rate limiting enzyme in glutathione biosynthesis. Two phylogenetically divergent species, mouse and the fruitfly, Drosophila melanogaster were used to test the hypothesis that reversible protein phosphorylation and pyridine dinucleotide phosphate dependent allostery regulate GCL activity. GCL was almost completely inhibited under phosphorylating conditions, involving preincubations with MgATP and endogenous protein kinases. Maximal GCL inhibitions of 94%, 77%, 85%, 87%, 83%, 95% and 89% occurred, respectively, in mouse cerebellum, hippocampus, brainstem, striatum, cortex and heart, and Drosophila. These changes in GCL activity were detected using saturating levels of substrates, suggesting that V(max) was dramatically affected, whereas K(m) values showed no differences. In vitro activation of GCL, presumably due to dephosphorylation, was blocked by inhibitors of protein phosphatases, suggesting that GCL exists in vivo as a mixture of phosphorylated and dephosphorylated forms. The reversibility of the dephosphorylation-dependent activation was indicated by the time-dependent inactivation of the in vitro activated Drosophila GCL, by preincubation with MgATP. NADPH increased maximal GCL activity by up to 93%, whereas several other nucleotide analogues did not, thereby demonstrating specificity. Kinetic analysis using Hanes-Woolf replots of initial velocity data suggested that the NADPH-dependent stimulation of GCL activity is brought about by a change in the maximal activity, V(max), rather than changes in substrate affinity. Results of this study suggest that mechanisms of modulation of eukaryotic GCL enzymes may include specific binding of ligands such as pyridine dinucleotide phosphates and reversible protein phosphorylation.     

Differential regulation of Na(+),K(+)-ATPase and the Na(+)-coupled glucose transporter in hypertensive rat kidney

Several Na(+) transporters are functionally abnormal in the hypertensive rat. Here, we examined the effects of a high-salt load on renal Na(+),K(+)-ATPase and the sodium-coupled glucose transporter (SGLT1) in Dahl salt-resistant (DR) and salt-sensitive (DS) rats. The protein levels of Na(+),K(+)-ATPase and SGLT1 in the DS rat were the same as those in the DR rat, and were not affected by the high-salt load. In the DS rat, a high-salt load decreased Na(+),K(+)-ATPase activity, and this decrease coincided with a decrease in the apparent Mechaelis constant (K(m)) for ATP, but not with a change of maximum velocity (V(max)). On the contrary, a high-salt load increased SGLT1 activity in the DS rat, which coincided with an increase in the V(max) for alpha-methyl glucopyranoside. The protein level of phosphorylated tyrosine residues in Na(+),K(+)-ATPase was decreased by the high-salt load in the DS rat. The amount of phosphorylated serine was not affected by the high-salt load in DR rats, and could not be detected in DS rats. On the other hand, the amount of phosphorylated serine residues in SGLT1 was increased by the high-salt load. However, the phosphorylated tyrosine was the same for all samples. Therefore, we concluded that the high-salt load changes the protein kinase levels in DS rats, and that the regulation of Na(+),K(+)-ATPase and SGLT1 activity occurs via protein phosphorylation.     

Factors affecting hexose phosphorylation in Acetobacter xylinum

Fructose was oxidized and converted to cellulose by cells of Acetobacter xylinum grown on fructose or succinate, but not by cells grown on glucose. In resting fructose-grown cells, glucose strongly suppressed fructose utilization. Extracts obtained from fructose- or succinate-grown cells catalyzed the adenosine triphosphate (ATP)-dependent formation of the 6-phosphate esters of glucose and fructose, whereas glucose-grown cell extracts phosphorylated glucose but not fructose. Fructokinase and glucokinase activities were separated and partially purified from cells grown on glucose, fructose, or succinate. Whereas fructokinase phosphorylated fructose only, glucokinase was active towards glucose and less active towards mannose and glucosamine. The optimal pH for the fructokinase was 7.4 and for the glucokinase was 8.5. The K(m) values for the fructokinase were: fructose, 6.2 mm; and ATP, 0.83 mm. The K(m) values for the glucokinase were: glucose, 0.22 mm; and ATP, 4.2 mm. Fructokinase was inhibited by glucose, glucosamine, mannose, and deoxyglucose in a manner competitive with respect to fructose, with K(i) values of 0.1, 0.14, 0.5, and 7.5 mm, respectively. Adenosine diphosphate (ADP) and adenosine monophosphate (AMP) inhibited both kinases noncompetitively with respect to ATP. The K(i) values were: 1.8 mm (ADP) and 2.1 mm (AMP) for fructokinase, and 2.2 mm (ADP) and 9.6 mm (AMP) for glucokinase. Fructose metabolism in A. xylinum appears to be regulated by the synthesis and activity of fructokinase.     

Enzymatic activity and substrate specificity of mitogen-activated protein kinase p38alpha in different phosphorylation states

The mitogen-activated protein (MAP) kinases are essential signaling molecules that mediate many cellular effects of growth factors, cytokines, and stress stimuli. Full activation of the MAP kinases requires dual phosphorylation of the Thr and Tyr residues in the TXY motif of the activation loop by MAP kinase kinases. Down-regulation of MAP kinase activity can be initiated by multiple serine/threonine phosphatases, tyrosine-specific phosphatases, and dual specificity phosphatases (MAP kinase phosphatases). This would inevitably lead to the formation of monophosphorylated MAP kinases. However, the biological functions of these monophosphorylated MAP kinases are currently not clear. In this study, we have prepared MAP kinase p38alpha, a member of the MAP kinase family, in all phosphorylated forms and characterized their biochemical properties. Our results indicated the following: (i) p38alpha phosphorylated at both Thr-180 and Tyr-182 was 10-20-fold more active than p38alpha phosphorylated at Thr-180 only, whereas p38alpha phosphorylated at Tyr-182 alone was inactive; (ii) the dual-specific MKP5, the tyrosine-specific hematopoietic protein-tyrosine phosphatase, and the serine/threonine-specific PP2Calpha are all highly specific for the dephosphorylation of p38alpha, and the dephosphorylation rates were significantly affected by different phosphorylated states of p38alpha; (iii) the N-terminal domain of MPK5 has no effect on enzyme catalysis, whereas deletion of the MAP kinase-binding domain in MKP5 leads to a 370-fold decrease in k(cat)/K(m) for the dephosphorylation of p38alpha. This study has thus revealed the quantitative contributions of phosphorylation of Thr, Tyr, or both to the activation of p38alpha and to the substrate specificity for various phosphatases.     

Molecular basis for substrate specificity of protein kinases and phosphatases

Regulation of various metabolic processes occurs by the phosphorylation/dephosphorylation of enzymes. Both the protein kinases that catalyze the phosphorylations and the protein phosphatases that catalyze the dephosphorylations display relatively broad specificity, reacting with a number of distinct sites in target enzymes. In this way changes in the activity of a particular kinase or phosphatase can cause coordinated and pleiotropic responses. However, the kinases and phosphatases do not exhibit a one-to-one correspondence in their reactions. Residues at different positions may be phosphorylated by a single kinase, yet dephosphorylated by different individual phosphatases. Conversely, sites which are substrates for different individual kinases may be dephosphorylated by a single phosphatase. In exploring the molecular basis for these differences this article shows that whereas kinases react with specific primary structures that often times appear as beta bends, the phosphatases recognize higher order structure, less strictly ruled by amino acid sequence surrounding the phosphorylated site. The differences, seen in the ability of these enzymes to utilize synthetic peptide substrates, might be rationalized in terms of function. Kinases need protruding segments of structure that can be enwrapped to exclude water, thereby minimizing ATP hydrolysis and enhancing phosphotransferase activity. On the other hand phosphatases are hydrolytic enzymes that may operate especially well on protein interfaces. Hydrolytic action often measured with p-nitrophenylphosphate is not necessarily indicative of a protein phosphatase and consideration of the mechanism reveals why this substrate can be misleading.(ABSTRACT TRUNCATED AT 250 WORDS)     

Probing the nucleotide binding and phosphorylation by the histidine kinase of a novel three-protein two-component system from Mycobacterium tuberculosis

The two-component signal transduction system from Mycobacterium tuberculosis bears a unique three-protein system comprising of two putative histidine kinases (HK1 and HK2) and one response regulator TcrA. By sequence analysis, HK1 is found to be an adenosine 5'-triphosphate (ATP) binding protein, similar to the nucleotide-binding domain of homologous histidine kinases, and HK2 is a unique histidine containing phosphotransfer (HPt)-mono-domain protein. HK1 is expected to interact with and phosphorylate HK2. Here, we show that HK1 binds 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate monolithium trisodium salt and ATP with a 1:1 stoichiometric ratio. The ATPase activity of HK1 in the presence of HK2 was measured, and phosphorylation experiments suggested that HK1 acts as a functional kinase and phosphorylates HK2 by interacting with it. Further phosphorylation studies showed transfer of a phosphoryl group from HK2 to the response regulator TcrA. These results indicate a new mode of interaction for phosphotransfer between the two-component system proteins in bacteria.     

Long chain fatty acids inhibit and medium chain fatty acids activate mammalian cardiac hexokinase

We investigated the effect of non-esterified fatty acids (FAs) on bovine heart hexokinase (type I: ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1). Long chain FAs (C14 to C20) inhibited the enzyme in a way that correlated positively with both the chain length and the degree of unsaturation. Medium chain FA with 12 or less carbons activated hexokinase in a chain length dependent manner with the greater activation shown by laurate. The activation constant of laurate was 91.5 microM with a maximal activation of 60.3%. Oleate caused a maximal decrease in specific activity of 25% with an inhibition constant of 79 microM. Using the fluorescent probe cis-parinarate, we found a saturable binding site with K(d) of 3.5 microM. Oleate competed the fluorescent probe from the protein with a K(d) of 1.4 microM. Medium chain FAs did not compete the probe from HK. The binding of fatty acid to the protein appears to be entropically driven as indicated by an Arrhenius analysis (DeltaS=+231.6 J mol(-1) deg(-1)). The presence of oleate significantly increased the K(ATP)(m) from 0.47 mM to 0.89 mM while the K(glucose)(m) in the presence of the FA (0.026+/-0.003 mM) was not significantly different from the control (0.014+/-0.004 mM). A decrease in V(max) values in the presence of oleate indicated that a mixed allosteric inhibition was operating.     

Dephosphorylation of Microtubule Proteins by Brain Protein Phosphatases 1 and 2A, and Its Effect on Microtubule Assembly

Protein phosphatase C was purified 140-fold from bovine brain with 8% yield using histone H1 phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase (cyclic AMP-kinase). Brain protein phosphatase C was considered to consist of 10 and 90%, respectively, of the catalytic subunits of protein phosphatases 1 and 2A on the basis of the effects of ATP and inhibitor-2. Protein phosphatase C dephosphorylated microtubule-associated protein 2 (MAP2), tau factor, and tubulin phosphorylated by a multifunctional Ca2+/calmodulin-dependent protein kinase (calmodulin-kinase) and the catalytic subunit of cyclic AMP-kinase. The properties of dephosphorylation of MAP2, tau factor, and tubulin were compared. The Km values were in the ranges of 1.6-2.7 microM for MAP2 and tau factor. The Km value for tubulin decreased from 25 to 10-12.5 microM in the presence of 1.0 mM Mn2+. No difference in kinetic properties of dephosphorylation was observed between the substrates phosphorylated by the two kinases. Protein phosphatase C did not dephosphorylate the native tubulin, but universally dephosphorylated tubulin phosphorylated by the two kinases. The holoenzyme of protein phosphatase 2A from porcine brain could also dephosphorylate MAP2, tau factor, and tubulin phosphorylated by the two kinases. The phosphorylation of MAP2 and tau factor by calmodulin-kinase separately induced the inhibition of microtubule assembly, and the dephosphorylation by protein phosphatase C removed its inhibitory effect. These data suggest that brain protein phosphatases 1 and 2A are involved in the switch-off mechanism of both Ca2+/calmodulin-dependent and cyclic AMP-dependent regulation of microtubule formation.     

Characterization of the ATP transporter in the reconstituted rough endoplasmic reticulum proteoliposomes

Adenosine triphosphate (ATP) transporter from rat liver rough endoplasmic reticulum (RER) was solubilized and reconstituted into phosphatidylcholine liposomes. The RER proteoliposomes, resulting from optimizing some reconstitution parameters, had an apparent K(m) value of 1.5 microM and a V(max) of 286 pmol min(-1) (mg protein)(-1) and showed higher affinity for ATP and a lower V(max) value than intact RER (K(m) of 6.5 microM and V(max) of 1 nmol). ATP transport was time- and temperature-dependent, inhibited by 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid, which is known as an inhibitor of anion transporters including ATP transporter, but was not affected by atractyloside, a specific inhibitor of mitochondrial ADP/ATP carrier. The internal and external effects of various nucleotides on the ATP transport were examined. ATP transport was cis-inhibited strongly by ADP and weakly by AMP. ADP-preloaded RER proteoliposomes showed a specific increase of ATP transport activity while AMP-preloaded RER proteoliposomes did not show the enhanced overshoot peak in the ATP uptake plot. These results demonstrate the ADP/ATP antiport mechanism of ATP transport in rat liver RER.     

Phosphorylated TandeMBP: A unique protein substrate for protein phosphatase assay

To analyze a variety of protein phosphatases, we developed phosphorylated TandeMBP (P-TandeMBP), in which two different mouse myelin basic protein isoforms were fused in tandem, as a protein phosphatase substrate. P-TandeMBP was prepared efficiently in four steps: (1) phosphorylation of TandeMBP by a protein kinase mixture (Ca²⁺/calmodulin-dependent protein kinase Iδ, casein kinase 1δ, and extracellular signal-regulated kinase 2); (2) precipitation of both P-TandeMBP and protein kinases to remove ATP, Pi, and ADP; (3) acid extraction of P-TandeMBP with HCl to remove protein kinases; and (4) neutralization of the solution that contains P-TandeMBP with Tris. In combination with the malachite green assay, P-TandeMBP can be used to detect protein phosphatase activity without using radioactive materials. Moreover, P-TandeMBP served as an efficient substrate for PPM family phosphatases (PPM1A, PPM1B, PPM1D, PPM1F, PPM1G, PPM1H, PPM1K, and PPM1M) and PPP family phosphatase PP5. Various phosphatase activities were also detected with high sensitivity in gel filtration fractions from mouse brain using P-TandeMBP. These results indicate that P-TandeMBP might be a powerful tool for the detection of protein phosphatase activities.     

A kinetic approach for the study of protein phosphatase-catalyzed regulation of protein kinase activity

The activities of many protein kinases are regulated by phosphorylation. The phosphorylated protein kinases thus represent an important class of substrates for protein phosphatases. However, our ability to study the phosphatase-catalyzed substrate dephosphorylation has been limited in many cases by the difficulty in preparing sufficient amount of stoichiometrically phosphorylated kinases. We have applied the kinetic theory of substrate reaction during irreversible modification of enzyme activity to the study of phosphatase-catalyzed regulation of kinase activity. As an example, we measured the effect of the hematopoietic protein-tyrosine phosphatase (HePTP) on the reaction catalyzed by the fully activated, bisphosphorylated extracellular signal-regulated protein kinase 2 (ERK2/pTpY). Because only a catalytic amount of ERK2/pTpY is required, this method alleviates the need for large quantities of phospho-ERK2. Kinetic analysis of the ERK2/pTpY-catalyzed substrate reaction in the presence of HePTP leads to the determination of the rate constants for the HePTP-catalyzed dephosphorylation of free ERK2/pTpY and ERK2/pTpY*substrate(s) complexes. The data indicate that ERK2/pTpY is a highly efficient substrate for HePTP (k(cat)/K(m) = 3.05 x 10(6) M(-1) s(-1)). The data also show that binding of ATP to ERK2/pTpY has no effect on ERK2/pTpY dephosphorylation by HePTP. In contrast, binding of an Elk-1 peptide substrate to ERK2/pTpY completely blocks the HePTP action. This result indicates that phosphorylation of Tyr185 is important for ERK2 substrate recognition and that binding of the Elk-1 peptide substrate to ERK2/pTpY blocks the accessibility of pTyr185 to HePTP for dephosphorylation. Collectively, the results establish that the kinetic theory of irreversible enzyme modification can be applied to study the phosphatase catalyzed regulation of kinase activity.     

The activity of the extracellular signal-regulated kinase 2 is regulated by differential phosphorylation in the activation loop

The mitogen-activated protein kinases (MAP kinases) play a central role in signaling pathways initiated by extracellular stimuli such as growth factors, cytokines, and various forms of environmental stress. Full activation of the MAP kinases requires dual phosphorylation of the Thr and Tyr residues in the TXY motif of the activation loop by MAP kinase kinases. Interestingly, down-regulation of MAP kinase activity can be initiated by multiple Ser/Thr phosphatases, Tyr-specific phosphatases, and dual-specificity phosphatases. This would inevitable lead to the formation of monophosphorylated MAP kinases. However, in much of the literature investigating MAP kinase signaling, there has been the implicit assumption that the monophosphorylated forms are inactive. Thus, the significance for the need of multiple phosphatases in regulating MAP kinase activity is not clear, and the biological functions of these monophosphorylated MAP kinases are currently unknown. We have prepared extracellular signal-regulated protein kinase 2 (ERK2) in all phosphorylated forms and kinetically characterized them using two proteins (the myelin basic protein and Elk-1) and ATP as substrates. Our results revealed that a single phosphorylation in the activation loop of ERK2 produces an intermediate activity state. Thus, the catalytic efficiencies of the monophosphorylated ERK2/pY and ERK2/pT (ERK2 phosphorylated on Tyr-185 and Thr-183, respectively) are approximately 2-3 orders of magnitude higher than that of the unphosphorylated ERK2 and are only 1-2 orders of magnitude lower than that of the fully active bisphosphorylated ERK2/pTpY. This raises the possibility that the monophosphorylated ERK2s may have distinct biological roles in vivo. Different phosphorylation states in the activation loop could be linked to graded effects on a single ERK2 function. Alternatively, they could be linked to distinct ERK2 functions. Although less active than the bisphosphorylated species, the monophosphorylated ERK2s may differentially phosphorylate pathway components.     

Protein kinase activity of rat brain hexokinase

Hexokinase 1 (HK1) purified from rat brain exhibits protein kinase activity, including autophosphorylation and phosphorylation of histone H2A. This protein kinase activity is observed only in the absence of the HK1 carbohydrate substrate, glucose. Analysis of the ATP-binding domains of the mammalian HK1 protein sequences shows significant homology with other mammalian protein kinases.     

A low molecular weight protein tyrosine phosphatase from Synechocystis sp. strain PCC 6803: enzymatic characterization and identification of its potential substrates

The predicted protein product of open reading frame slr0328 from Synechocystis sp. PCC 6803, SynPTP, possesses significant amino acid sequence similarity with known low molecular weight protein tyrosine phosphatases (PTPs). To determine the functional properties of this hypothetical protein, open reading frame slr0328 was expressed in Escherichia coli. The purified recombinant protein, SynPTP, displayed its catalytic phosphatase activity towards several tyrosine, but not serine, phosphorylated exogenous protein substrates. The protein phosphatase activity of SynPTP was inhibited by sodium orthovanadate, a known inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor for many serine/threonine phosphatases. Kinetic analysis indicated that the K(m) and V(max) values for SynPTP towards p-nitrophenyl phosphate are similar to those of other known bacterial low molecular weight PTPs. Mutagenic alteration of the predicted catalytic cysteine of PTP, Cys(7), to serine abolished enzyme activity. Using a combination of immunodetection, mass spectrometric analysis and mutagenically altered Cys(7)SerAsp(125)Ala-SynPTP, we identified PsaD (photosystem I subunit II), CpcD (phycocyanin rod linker protein) and phycocyanin-α and -β subunits as possible endogenous substrates of SynPTP in this cyanobacterium. These results indicate that SynPTP might be involved in the regulation of photosynthesis in Synechocystis sp. PCC 6803.     

Regulation of glucose-6-phosphate dehydrogenase by reversible phosphorylation in liver of a freeze tolerant frog

Glucose-6-phosphate dehydrogenase (G6PDH) and the pentose phosphate pathway play a key role in reductive biosynthesis and antioxidant defense, while diverting glucose from other cellular functions. G6PDH was isolated from liver of the wood frog, Rana sylvatica, a freeze tolerant species that uses glucose as a cryoprotectant. Analysis of kinetic parameters (K _m and V _max) of G6PDH showed a significant increase in K _m G6P (from 98.2 ± 3.8 to 121 ± 5.3 μM) and K _m NADP⁺ (from 65.5 ± 2.3 to 89.1 ± 4.8 μM) in frogs following freezing exposure, indicating lower affinity for G6PDH substrates in this state. Subsequent analyses indicated that differential phosphorylation of G6PDH between the two states was responsible for the altered kinetic properties. Thus, two differentially charged forms of G6PDH were resolved by DEAE ion-exchange chromatography and, compared with controls, the proportion of G6PDH activity in peak I decreased and in peak II increased in liver from frozen frogs. G6PDH in peak I had a K _m G6P of 94.1 ± 1.1 μM and K _m NADP⁺ of 61.2 ± 3.5 μM, whereas Peak II G6PDH showed higher values (K _m G6P was 172 ± 4.3 μM, K _m NADP⁺ was 98.2 ± 3.3 μM). G6PDH from each peak was incubated with ions and second messengers to stimulate the actions of protein kinases with results indicating that G6PDH can be phosphorylated by protein kinase G, protein kinase C, AMP-activated protein kinase, or calmodulin-dependent protein kinase. The data indicate that in control frogs, G6PDH is in a high phosphate form and displays a high substrate affinity, whereas in frozen frogs G6PDH is less phosphorylated, with lower substrate affinity.