Role of the deinhibitor protein in the interconversion of the ATP,Mg-dependent protein phosphatase

The small molecular weight (± 9,000) heat stable deinhibitor protein, isolated from dog liver, not only protects the multisubstrate protein phosphatase from inhibition by inhibitor-1 and the modulator protein. It prevents the conversion of the active enzyme to the ATP,Mg-dependent enzyme form brought about by the modulator protein, and also affects the activation of the ATP,Mg-dependent protein phosphatase, probably by stabilizing the enzyme in its active conformation during the reversible activation by protein kinase F_A. Therefore the deinhibitor protein could be an important factor in the process of glycogen synthesis, which requires glycogen synthase and phosphorylase as dephosphorylated enzymes.     

Inactivation of I(A) channels of frog skeletal muscle is modulated by ATP

Whole cell, voltage clamp experiments were performed in vesicles derived from frog skeletal muscle plasma membranes to characterize the influence of ATP on the kinetic properties of fast inactivating K(+) currents (I(A)). I(A) was recorded in ATP-free solutions. Peak I(A) decayed with a time constant of 27 ms at large depolarizations. Steady state inactivation reached half maximal values at -66 mV. In the presence of ATP, these values were 196 ms and -41 mV, respectively, indicating a major effect of ATP on inactivation. In contrast, activation of I(A) was unaffected by ATP. The protein kinase C (PKC) inhibitors, H7 and staurosporine, greatly prevented the effects of ATP on inactivation. Inactivation remained unchanged by the protein kinase A inhibitor HA1004 or by the catalytic subunit of cAMP protein kinase. We conclude that ATP decreases inactivation of skeletal muscle I(A) and that this effect may be mediated by protein kinase C.     

Activation of the ATP.Mg-dependent type 1 protein phosphatase by the Fe2+/ascorbate system

The ATP.Mg-dependent type 1 protein phosphatase is inactive as isolated but can be activated in several different ways. In this report, we show that the phosphatase can also be activated by the Fe²⁺/ascorbate system. Activation of the phosphatase requires both Fe²⁺ ion and ascorbate and the level of activation is dependent on the concentrations of Fe²⁺ ion and ascorbate. In the presence of 20 mM ascorbate, the Fe²⁺ ion concentrations required for half-maximal and maximal activation are about 0.3 and 3mM, respectively. Several common divalent metal ions, including Co²⁺, Ni²⁺, Cu²⁺, Mg²⁺, and Ca²⁺ ions, cannot cooperate with ascorbate to activate the phosphatase, and SH-containing reducing agents such as 2-mercaptoethanol and dithiothreitol cannot cooperate with Fe²⁺ ion to activate the phosphatase, indicating that activation of the phosphatase by the Fe²⁺/ascorbate system is a specific process. Moreover, H₂O₂, a strong oxidizer, could significantly diminish the phosphatase activation by the Fe²⁺/ascorbate system, suggesting that reduction mechanism other than SH-SS interchange is a prerequisite for the Fe²⁺/ascorbate-mediated phosphatase activation. Taken together, the present study provides initial evidence for a new mode of type 1 protein phosphatase activation mechanism.Abbreviations MAPK mitogen-activated protein kinase - MCO metal ion-catalyzed oxidation - kinase F_A the activating factor of ATP.Mg-dependent protein phosphatase - I₂ inhibitor-2 - EDTA ethylenediaminetetraacetic acid - MBP myelin basic protein     

Immunological and biochemical study on tissue and subcellular distributions of protein kinase FA (an activating factor of ATP.Mg-dependent protein phosphatase): A simplified and efficient procedure for high quantity purification from brain

Although protein kinase F_A/GSK-3 (an activating factor of ATP.Mg-dependent protein phosphatase) has been established as a cytosolic enzyme in mammalian nonnervous tissues involved in the metabolic regulation, immunological and biochemical studies on tissue and subcellular distributions demonstrate that kinase F_A/GSK-3 is in fact a membrane-associated enzyme and most abundantly exists in brain particulate membrane fractions depending on the tissue homogenization conditions. For instance, when brain was homogenized in Polytron without 0.32M sucrose, approximately 40% of the total F_A/GSK-3 was found in the cytosol. However, when brain was homogenized in buffer containing 0.32M sucrose and in a glass homogenizer with Teflon pestle, more than 80% of the total F_A/GSK-3 was found associated with the particulate membrane fractions. By manipulating these findings, we have developed a simplified procedure for purification of homogeneous kinase F_A/GSK-3 in high recovery and in a substantial amount from brain tissue. The data explain why kinase F_A/GSK-3 cannot be isolated in a reasonable amount from most mammalian tissues for the past years. The specific pure antibody that can specifically recognize kinase F_A/GSK-3 from crude tissue extracts together with the high quantity purification of the enzyme as presented in this report provides an initial key step for studies on the role of kinase F_A/GSK-3 in the regulation of brain functions especially in the brain particulate membrane fractions.     

Regulatory effects of S-100 protein and parvalbumin on protein kinases and phosphoprotein phosphatases from brain and skeletal muscle

Summary In the eluted fractions of histone-treated crude extracts separated by Sephadex G-200 filtration, multiple protein kinase (PK) activities, including three from brain and two from skeletal muscle, were augmented by both S-100 protein and parvalbumin on the phosphorylation of endogenous substrates. One additional PK activity suppressed by both S-100 and parvalbumin was also found in muscle. In comparison, phosphoprotein phosphatases (PPase), which were also prepared by the same procedure of initial step of histone-treatment followed by the steps of Bio-Gel P-6DG for brain and DNA-cellulose for muscle, were all activated by S-100 while inhibited by parvalbumin and phosphatidylserine.     

On the mechanism of activation of protein kinase FA (an activating factor of ATP.Mg-dependent protein phosphatase) in brain myelin

Protein kinase F_A (an activating factor of ATP·Mg-dependent protein phosphatase) has been characterized to exist in two forms in the purified brain myelin. One form of kinase F_A is spontaneously active and trypsin-labile, whereas the other form of kinase F_A is inactive and trypsin-resistant, suggesting a different membrane topography with active F_A exposed on the outer face of the myelin membrane and inactivu F_Q buried within the myelin membrane. When myelin was solubilized in 1% Triton X-100, all kinase F_A became active and trypsin-labile. Phospholipid reconstitution studies further indicated that when kinase F_A was reconstituted in acidic phospholipids, such as phosphatidylinositol and phosphatidylserine, the enzyme activity was inhibited in a dose-dependent manner, suggesting that kinase F_A interacts with acidic phospholipids which inhibit its activity. Furthermore, when myelin was incubated with exogenous phospholipase C, the inactive/trypsin-resistant F_A could be converted to the active/trypsin-labile F_A in a time- and dose-dependent manner. Taken together, it is concluded that membrane phospholipids play an important role in modulating the activity of kinase F_A in the brain myelin. It is suggested that phospholipase C may mediate the activation-sequestration of inactive/trypsin-resistant kinase F_A in the brain myelin through the phospholipase C-katalyzed degradation of acidic membrane phospholipids. The activation-sequestration of protein Kinase F_A may represent one mode of control modulating the activity of kinase F_A in the central nervous system myelin.     

Identification and characterization of DUSP27, a novel dual-specific protein phosphatase

A novel human dual-specific protein phosphatase (DSP), designated DUSP27, is here described. The DUSP27 gene contains three exons, rather than the predicted 4-14 exons, and encodes a 220 amino acid protein. DUSP27 is structurally similar to other small DSPs, like VHR and DUSP13. The location of DUSP27 on chromosome 10q22, 50 kb upstream of DUSP13, suggests that these two genes arose by gene duplication. DUSP27 is an active enzyme, and its kinetic parameters and were determined. DUSP27 is a cytosolic enzyme, expressed in skeletal muscle, liver and adipose tissue, suggesting its possible role in energy metabolism.     

Support for a trimeric collar of myosin binding protein C in cardiac and fast skeletal muscle, but not in slow skeletal muscle

Myosin-binding protein C (MyBPC) is proposed to take on a trimeric collar arrangement around the thick filament backbone in cardiac muscle, based on interactions between cardiac MyBPC domains C5 and C8. We have now determined, using yeast two-hybrid and in vitro binding assays, that the C5:C8 interaction is not dependent on the 28-residue cardiac-specific insert in C5. Furthermore, an interaction of similar affinity occurs between domains C5 and C8 of fast skeletal muscle MyBPC, but not between these domains of the slow skeletal muscle protein. These data have implications for the role and quaternary structure of MyBPC in skeletal muscle.     

Purification,characterization and partial amino acid sequencing of a 70 kD inhibitor protein of Na+,K+-ATPase from goat testis cytosol

A protein isolated from goat testis cytosol is found to inhibit Na⁺,K⁺-ATPase from rat brain microsomes. The inhibitor has been purified by ammonium sulphate precipitation followed by hydroxyapatite column chromatography. The purified fraction appears as a single polypeptide band on 10% SDS-PAGE of approximate molecular mass of 70 kDa. The concentration at which 50% inhibition (I₅₀) occurs is in the nanomolar range. The inhibitor seems to bind Na⁺,K⁺-ATPase reversibly at ATP binding site in a competitive manner with ATP, but away from ouabain binding site. It does not affect p-nitrophenyl-phosphatase activity. The inhibitor is found to inhibit the phosphorylation step of the Na⁺,K⁺-ATPase. The enhancement of tryptophan fluorescence and changes in CD pattern suggest conformational changes of Na⁺,K⁺-ATPase on binding to the inhibitor. Amino acid sequence of the trypsinised fragments show some homology with aldehyde reductase.     

ATP x Mg-dependent protein phosphatase from rabbit skeletal muscle. II. Purification of the activating factor and its characterization as a bifunctional protein also displaying synthase kinase activity

A protein (FA) has been isolated from rabbit muscle which has two functions: one is the activation of the ATP x Mg-dependent phosphatase (see previous paper) (1) and the second is the phosphorylation and concomitant inactivation of glycogen synthase, independent from cyclic AMP or Ca ions. The two activities co-purify throughout the purification scheme, and reside in the single protein band that the purified preparation shows in discontinuous acrylamide gel electrophoresis. Heat inactivation experiments with the purified protein showed a parallel decrease of both activities with time. GTP could efficiently replace the ATP in both reactions. Sodium dodecyl sulfate-gel electrophoresis also shows a single protein-stained band corresponding to a Mr = approximately 50,000 and sucrose density gradient centrifugation gave a value of 45,000. The enzyme incorporates only 1 mol of phosphate/mol of synthase monomer (85,000 daltons) and brings the activity ratio (+/- glucose-6-P) down to less than 0.05. Kinetic studies suggest that FA exerts its two activities in quite different ways: the activation of the ATP x Mg-dependent phosphatase is bought about by a protein-protein interaction (FA x FC complex formation) with ATP x Mg as a necessary cofactor, whereas for the inactivation of synthase, FA is a cyclic AMP- and Ca-independent kinase.     

Effect of proline on thermal inactivation,denaturation and aggregation of glycogen phosphorylase b from rabbit skeletal muscle

It has been shown that the relatively low concentrations of proline (0.1 M) have a slight accelerating effect on thermal aggregation of glycogen phosphorylase b (Phb) from rabbit skeletal muscle registered by the accumulaton of the aggregated protein. The suppression of Phb aggregation at high proline concentrations is mainly due to the protective action of proline on the stage of unfolding of the Phb molecule. The enhancement of Phb stability in the presence of the high concentrations of proline was demonstrated by the data on differential scanning calorimetry, analytical ultracentrifugation and thermoinactivation kinetics. The construction of the protein aggregate size versus time plots allowed the acceleration of the stage of Phb aggregation in the presence of high concentrations of proline to be demonstrated. The obtained results are consistent with the predictions of the crowding theory.     

IF1, a natural inhibitor of mitochondrial ATP synthase,is not essential for the normal growth and breeding of mice

IF1 is an endogenous inhibitor protein of mitochondrial ATP synthase. It is evolutionarily conserved throughout all eukaryotes and it has been proposed to play crucial roles in prevention of the wasteful reverse reaction of ATP synthase, in the metabolic shift from oxidative phosphorylation to glycolysis, in the suppression of ROS (reactive oxygen species) generation, in mitochondria morphology and in haem biosynthesis in mitochondria, which leads to anaemia. Here, we report the phenotype of a mouse strain in which IF1 gene was destroyed. Unexpectedly, individuals of this IF1-KO (knockout) mouse strain grew and bred without defect. The general behaviours, blood test results and responses to starvation of the IF1-KO mice were apparently normal. There were no abnormalities in the tissue anatomy or the autophagy. Mitochondria of the IF1-KO mice were normal in morphology, in the content of ATP synthase molecules and in ATP synthesis activity. Thus, IF1 is not an essential protein for mice despite its ubiquitous presence in eukaryotes.     

Thromboxane A2 stimulates mitogen activated protein kinase and arachidonic acid liberation in rabbit platelets

U46619, a thromboxane A₂ mimetic, caused tyrosine phosphorylation of several proteins in rabbit platelets. Among them, 42 kDa protein was identified as a mitogen-activated protein kinase (MAPK). U46619 activated MAPK in a concentration-dependent manner, measured by incorporation of ³²P to a specific substrate for MAPK. U46619 also liberated [³H)arachidonic acid in a concentration-dependent manner. The U46619-induced MAPK activation and [³H]arachidonic acid liberation were inhibited by SQ29548 and by the removal of external Ca²⁺ ions. This is a first demonstration that TXA₂ activates MAPK accompanied with arachidonic acid liberation in rabbit platelets.     

F0F1 ATP synthase of Streptomycetes: Modulation of activity and oligomycin resistance by protein Ser/Thr kinases

Inverted membrane vesicles of Gram-positive actinobacteria Streptomyces fradiae, S. lividans, and S. avermitilis have been prepared and membrane-bound F₀F₁ ATP synthase has been biochemically characterized. It has been shown that the ATPase activity of membrane-bound F₀F₁ complex is Mg²⁺-dependent and moderately stimulated by high concentrations of Ca²⁺ ions (10–20 mM). The ATPase activity is inhibited by N,N′-dicyclohexylcarbodiimide and oligomycin A, typical F₀F₁ ATPase inhibitors that react with the membrane-bound F₀ complex. The assay of biochemical properties of the F₀F₁ ATPases of Streptomycetes in all cases showed the presence of ATPase populations highly susceptible and insensitive to oligomycin A. The in vitro labeling and inhibitory assay showed that the inverted phospholipid vesicles of S. fradiae contained active membrane-bound Ser/Thr protein kinase(s) phosphorylating the proteins of the F₀F₁ complex. Inhibition of phosphorylation leads to decrease of the ATPase activity and increase of its susceptibility to oligomycin. The in vivo assay confirmed the enhancement of actinobacteria cell sensitivity to oligomycin after inhibition of endogenous phosphorylation. The sequencing of the S. fradiae genes encoding oligomycin-binding A and C subunits of F₀F₁ ATP synthase revealed their close phylogenetic relation to the genes of S. lividans and S. avermitilis.     

Fatty acid effects on calcium influx and efflux in sarcoplasmic reticulum vesicles from rabbit skeletal muscle

Low concentrations of fatty acids inhibited initial Ca uptake by sarcoplasmic reticulum vesicles, the extent of inhibition varying with chain length and unsaturation in a series of C₁₄–C₂₀ fatty acids. Oleic acid was a more potent inhibitor of initial Ca uptake than stearic acid at 25°C, whereas at 5°C there was less difference between the inhibitory effects of low concentrations of these fatty acids. When the fatty acids were added later, during the phase of spontaneous Ca release that follows Ca uptake in reactions carried out at 25°C, 1–4 μM oleic and stearic acids caused Ca content to increase. This effect was due to marked inhibition of Ca efflux and slight stimulation of Ca influx. At concentrations of >4 μM, both fatty acids inhibited the Ca influx that occurs during spontaneous Ca release; in the case of oleic acid, this inhibition resembled that of initial Ca uptake at 5°C. The different effects of fatty acids at various times during Ca uptake reactions may be explained in part if alterations in the physical state of the membranes occur during the transition from the phase of initial Ca uptake to that of spontaneous Ca release.     

Adaptive evolution of the vertebrate skeletal muscle sodium channel

Tetrodotoxin (TTX) is a highly potent neurotoxin that blocks the action potential by selectively binding to voltage-gated sodium channels (Na(v)). The skeletal muscle Na(v) (Na(v)1.4) channels in most pufferfish species and certain North American garter snakes are resistant to TTX, whereas in most mammals they are TTX-sensitive. It still remains unclear as to whether the difference in this sensitivity among the various vertebrate species can be associated with adaptive evolution. In this study, we investigated the adaptive evolution of the vertebrate Na(v)1.4 channels. By means of the CODEML program of the PAML 4.3 package, the lineages of both garter snakes and pufferfishes were denoted to be under positive selection. The positively selected sites identified in the p-loop regions indicated their involvement in Na(v)1.4 channel sensitivity to TTX. Most of these sites were located in the intracellular regions of the Na(v)1.4 channel, thereby implying the possible association of these regions with the regulation of voltage-sensor movement.     

Low resolution structure of subunit b (b (22-156)) of Escherichia coli F(1)F(O) ATP synthase in solution and the b-delta assembly

The first low resolution solution structure of the soluble domain of subunit b (b _22–156) of the Escherichia coli F₁F_O ATPsynthase was determined from small-angle X-ray scattering data. The dimeric protein has a boomerang-like shape with a total length of 16.2 ± 0.3 nm. Fluorescence correlation spectroscopy (FCS) shows that the protein binds effectively to the subunit δ, confirming their described neighborhood. Using the recombinant C-terminal domain (δ_91–177) of subunit δ and the C-terminal peptides of subunit b, b _120–140 and b _140–156, FCS titration experiments were performed to assign the segments involved in δ–b assembly. These data identify the very C-terminal tail b _140–156 to interact with δ_91–177. The novel 3D structure of this peptide has been determined by NMR spectroscopy. The molecule adopts a stable helix formation in solution with a flexible tail between amino acid 140 to 145.     

Acceptor activity, isoacceptor profiles and function in protein synthesis of transfer RNAs from regenerating skeletal muscle

Transfer RNAs have been prepared from control and regenerating rat skeletal muscle. The yield of tRNA is highest during the early stages of the regeneration process (5 and 8 days following the induction of regeneration) and decreases to near control values thereafter. The amino acid acceptor activity (extent of aminoacylation) of tRNA from regenerating muscle was also found to be higher for some amino acids than the activity of control tRNA, and the maximum increase in activity was observed between 5 and 8 days following the initiation of regeneration with a decrease to control levels through 15 and 30 days. The isoacceptor pattern, determined by RPC-5 chromatography, for methionyl-tRNAs from control muscle and 5-day regenerating muscle were essentially indistinguishable, while a minor peak of prolyl-tRNA was observed in the population from 5-, 8- and 15-day regenerates which was apparently absent from the control tRNA. Lysyl-tRNAs from control muscle contain two major isoacceptors while a third isoacceptor is observed in the tRNA preparations from 5-, 8- and 15-day regenerating muscle. The relative amount of this third isoacceptor is highest in the 8-day population and decreases in amount in tRNAs from 15- and 30-day regenerates. Control muscle also contains two major glutamyl-tRNA species while a third isoacceptor can be detected in regenerates. The relative amount of this species increases during the early course of the regeneration process but is present at near control levels by 30 days following Marcaine injection. Cell-free protein synthesis using muscle polyribosomes showed that tRNAs from regenerating muscle were more effective in stimulating [³⁵S]methionine incorporation than tRNAs from control muscle.     

Spectral and kinetic characterization of electron acceptor A1 in a Photosystem I core devoid of iron-sulfur centers FX,FB and FA

The kinetic and spectroscopic properties of the secondary electron acceptor A₁ were determined by flash absorption spectroscopy at room and cryogenic temperatures in a Photosystem I (PS I) core devoid of the iron-sulfur clusters F_X, F_B and F_A. It was shown earlier (Warren, P.V., Golbeck, J.H. and Warden, J.T. (1993) Biochemistry 32: 849–857) that the majority of the flash-induced absorbance increase at 820 nm, reflecting formation of P700⁺, decays with a t_1/2 of 10 s due to charge recombination between P700⁺ and A₁ ^–. Following A₁ ^– directly around 380 nm, where absorbance changes due to the formation of P700⁺ are negligible, two major decay components were resolved in this study with t_1/2 of 10 s and 110 s at an amplitude ratio of 2.5:1. The difference spectra between 340 and 490 nm of the two kinetic phases are highly similar, showing absorbance increases from 340 to 400 nm characteristic of the one-electron reduction of the phylloquinone A₁. When measured at 10 K, the flash-induced absorbance changes around 380 nm can be fitted with two decay phases of t_1/2 15 s and 150 s at an amplitude ratio 1:1. The difference spectra of both kinetic phases from 340 to 400 nm are similar to those determined at 298 K and are therefore attributed to charge recombination in the pair P700⁺A₁ ^–. These results indicate that the backreaction between P700⁺ and A₁ ^– is multiphasic when F_X, F_B and F_A are removed, and only slightly temperature dependent in the range of 298 K to 10 K.Abbreviations Chl chlorophyll - D pathlength for the measuring light through the sample - DPIP 2,6-dichlorophenolindophenol - EPR electron paramagnetic resonance - IR infrared - PS I Photosystem I - Tris Tris(hydroxymethyl)aminomethane - UV ultraviolet Published as Journal Series #10890 of the University of Nebraska Agricultural Research Division and supported by a grant from the National Science Foundation (MCB-9205756).