Substitution of a proline for alanine 183 in the hinge region of phosphoglycerate kinase: Effects on catalysis, activation by sulfate, and thermal stability

A hinge-bending domain movement has been postulated as an important part of the catalytic mechanism of phosphoglycerate kinase (PGK) (Bankset al., 1979). In order to test the role of the flexibility of a putative interdomain hinge in the substrate- and sulfate-induced conformational transitions, alanine-183 was replaced by proline using site-directed mutagenesis. The maximal velocity of the Ala 183Pro mutant, measured at saturating concentrations of ATP and phosphoglycerate (5 mM and 10 mM, respectively) and in the absence of sulfate ions, is increased approximately 21% in comparison to the wild type PGK. TheK _m values for both substrates are essentially unchanged. The effect of sulfate on the specific activity of the Ala 183Pro mutant and the wild type PGK was measured in the presence of 1 mM ATP and 2 mM 3-phosphoglycerate (3-PG). A maximum activation of 70% was observed at 20 mM sulfate for the mutant enzyme, as compared to 130% activation at 30 mM sulfate for the wild type PGK. These results demonstrate that the increased rigidity of the putative hinge, introduced by the AlaPro mutation, does not impair catalytic efficiency of phosphoglycerate kinase, while it appears to decrease the sulfate-dependent activation. The differential scanning calorimetry (DSC) studies demonstrate an increased susceptibility of the Ala 183 Pro mutant to thermal denaturation. In contrast to one asymmetric transition observed in the DSC scan for the wild type PGK, withT _m near 54°C, two transitions are evident for the mutant enzyme withT _m values of about 45 and 54°C. Using a thermodynamic model for two interacting domains, a decrease in the free energy of domain-domain interactions of about 2 kcal was estimated from the DSC data.     

Creatine kinase activity of urinary bladder and skeletal muscle from control and streptozotocin-diabetic rats

The urinary bladder depends on intracellular ATP for the support of a number of essential intracellular processes including contraction. The concentration of ATP is maintained constant primarily via the rapid transfer of a phosphate from creatine phosphate (CP) to ADP catalyzed by the enzyme creatine kinase (CK). Since muscular pathologies associated with diabetes are in part related to intracellular alterations in metabolism, we have characterized the CK activity in both skeletal muscle and urinary bladder from control and streptozotocin-diabetic rats.The following is a summary of the results: 1) Bladder tissue from control rats showed linear kinetics with a V_max = 390 nmoles/mg protein/min, and a K_m = 275 µM. 2) Urinary bladder tissue isolated from diabetic rats displayed biphasic kinetics with V_max = 65 and 324 nmoles/mg protein/min, and K_m's = 10 µM and 190 µM respectively. 3) Skeletal muscle isolated from control rats showed linear kinetics with an approximate V_max of 800 nmoles/mg protein/min and a K_m of 280 µM CP. 4) Homogenates of skeletal muscle from diabetic rats showed complex kinetics not separable into distict component forms. 5) The K_m for ADP for both skeletal muscle and bladder was approximately 10 µM.These studies demonstrate that whereas bladders isolated from both control and diabetic rats possess a low-affinity isomer(s) of CK with similar maximum enzymatic activity, there is a high affinity isomer present within the urinary bladder muscle of diabetic rats that is not present in bladder tissue isolated from control rats. Skeletal muscle isolated from both diabetic and control rats exhibited a maximal activity 2 to 3 times higher than that of the bladder.     

The Drosophila PROS-28.1 gene is a member of the proteasome gene family

In the present communication, we report the identification of a new gene family which encodes the protein subunits of the proteasome. The proteasome is a high-M_r complex possessing proteolytic activity. Screening a Drosophila λgt11 cDNA expression library with the proteasome-specific antibody N19-28 we isolated a clone encoding the 28-kDa No. 1 proteasome protein subunit. In accordance with the nomenclature of proteasome subunits in Drosophila, the corresponding gene is designated PROS-28.1, and it encodes an mRNA of 1.1 kb with an open reading frame of 249 amino acids (aa). Genomic Southern-blot hybridization shows PROS-28.1 to be a member of a family of related genes. Analysis of the predicted aa sequence reveals a potential nuclear targeting signal, a potential site for tyrosine kinase and a potential cAMP/cGMP-dependent phosphorylation site. The aa sequence comparison of the products of PROS-28.1 and PROS-35 with the C2 proteasome subunit of rat shows a strong sequence similarity between the different proteasome subunits. The data suggest that at least a subset of the proteasome-encoding genes belongs to a family of related genes (PROS gene family) which may have evolved from a common ancestral PROS gene.     

Protein phosphorylation in Streptomyces albus

The phosphorylated proteins of Streptomyces albus, radioactively labeled with [32P]orthophosphate have been analyzed by gel electrophoresis and autoradiography. More than 10 protein species were found to be phosphorylated. With [32P]ATP as substrate cell free extracts phosphorylated endogenous proteins in vitro which were predominantly phosphorylated in vivo. From cell extract which exhibited active phosphorylated in vitro, a protein kinase has been partially purified. The kinase activity was identified in fractions corresponding to a 90 kDa protein.     

Suppression of protein kinase C and the stimulation of glucocorticoid receptor synthesis by dexamethasone in human fibroblasts derived from tumor tissue

Exposure of fibroblasts derived from keloid tissues, desmoid and dermal tissue from individuals with Gardner's syndrome (GS) to dexamethasone resulted in the suppression of protein kinase C (PKC) activity and [3H]thymidine incorporation into DNA, and a 20-fold induction of glutamine synthetase activity. Treatment of GS and keloid fibroblasts with 0.1 microM dexamethasone for 36 h increased glucocorticoid receptor (GR) synthesis, as determined by [35S]methionine labeling and immunoprecipitation with a monoclonal antibody to the human GR. The suppression of PKC activity by dexamethasone was shown to result from a loss of protein mass as determined by immunoblotting using an antibody to PKC type III. In contrast to these results, exposure of fibroblasts isolated from normal tissues to dexamethasone did not result in the suppression PKC and [3H]thymidine incorporation, there was only a sixfold induction of glutamine synthetase, and a decrease of GR synthesis. As no primary receptor binding defect could be detected, the altered response of tumor cells to steroid-occupied receptor indicates a partial post-receptor binding defect in GS and keloid cells.     

Circadian Variations in the Affinities of Nad Kinase and Nadp Phosphatase for their Substrates, Nad+ and Nadp+ in Dividing and Nondividing Cells of the Achlorophyllous Zc Mutant of Euglena Gracilis Klebs (Strain Z)

-We have previously shown that NAD kinase and NADP phosphatase activities display circadian rhythms, in the soluble (SN) and membrane-bound (P) fractions of crude extracts of the achlorophyllous ZC mutant of the phytoflagellate Euglena gracilis (which displays circadian rhythmicity of cell division). We determined if changes in the affinity of NADP phosphatase and NAD kinase for their substrates, NADP⁺ and NAD⁺, were occurring by calculating the ratios 100(velocity found in Km conditions/velocity found in saturating conditions). The rationale was that if the affinity remained unchanged according to circadian time (CI), these values should always equal 50, independently of any changes in enzyme quantity; values greater than 50 should indicate increases in enzyme affinity, and values less than 50 decreases in affinity. Our results indicated that these values calculated for NADP phosphatase exhibited a complex pattern of rhythmicity, while those for NAD kinase displayed circadian variations strongly correlated with the rhythms in enzyme activity. The curves showed troughs at CT 00-04 both in dividing and nondividing cells and peaks at CT 18-20 or at CT 08-14 in cells sampled, respectively, from a dividing or a stationary culture. Such variations are indicative of changes in the kinetic properties of the enzyme, which may reflect modifications in its affinity either for effectors (such as Ca2⁺-calmodulin) or for its substrate, NAD⁺. This may be due to (i) the expression of different isoenzymes at different CTs; (ii) different posttranslational modifications of the enzyme; or (iii) concentrations of effectors varying in a circadian manner.     

Hormonally regulated expression of arginine kinase in Drosophila melanogaster

Summary Arginine kinase (AK) is present throughout the life cycle of Drosophila melanogaster, but there is a sharp, transient peak of AK activity during the prepupal period and a second period of elevated activity at the time of eclosion of the adult. Imaginal discs show the greatest increase in AK activity at the prepupal stage of those tissues assayed. The prepupal peak is not seen when the temperature-sensitive ecdysoneless mutant ecd-1 is shifted to 29° C at mid-third instar larval stage. The peak in activity reappears when ecd-1 is either shifted back to 20° C after 60 h at 29° C or is fed 20-hydroxyecdysone. At the restrictive temperature, imaginal discs from ecd-1 larvae progressively lose AK activity, whereas discs from 20-hydroxyecdysone-fed larvae have a marked increase in AK activity at stage P3 of the prepupal period. These data suggest that the prepupal peak is regulated by the hormone 20-hydroxyecdysone.     

The role of protein kinases and protein phosphatases in the regulation of cardiac sarcoplasmic reticulum function

Summary Canine cardiac sarcoplasmic reticulum is phosphorylated by adenosine 3,5-monophosphate (cAMP)-dependent and by calcium · calmodulin-dependent protein kinases on a 27 000 proteolipid, called phospholamban. Both types of phosphorylation are associated with an increase in the initial rates of Ca²⁺ transport by SR vesicles which reflects an increased turnover of elementary steps of the calcium ATPase reaction sequence. The stimulatory effects of the protein kinases on the calcium pump may be reversed by an endogenous protein phosphatase, which can dephosphorylate both the CAMP-dependent and the calcium · calmodulin-dependent sites on phospholamban. Thus, the calcium pump in cardiac sarcoplasmic reticulum appears to be under reversible regulation mediated by protein kinases and protein phosphatases.     

Post-receptor modulation of the effects of cyclic AMP in isolated cardiac myocytes

Summary The actions of cyclic AMP are subject to several levels of post-receptor modulation in cardiac tissue. Isoproterenol and prostaglandin E₁ both stimulate cAMP accumulation, but only isoproterenol causes activation of particulate cAMP-dependent protein kinase, leading to activation of phosphorylase kinase and glycogen phosphorylase, and inhibition of glycogen synthase. Through the use of isolated, adult ventricular myocytes, we have determined that the hormone-specific activation of glycogen phosphorylase is due to subcellular compartmentation of cAMP. There is some evidence that cyclic nucleotide phosphodiesterases, whose activity is stimulated by alpha₁-adrenergic agonists in isolated myocytes, may have a role in compartmentation. Phosphoinositide hydrolysis is stimulated by alpha, and muscarinic agonists, presumably leading to activation of protein kinase C, which in turn has multiple effects on hormone-sensitive adenylate cyclase.Abbreviations cAMP Adenosine-3,5-Cyclic Monophosphate - cGMP Guanosine-3,5-Cyclic Monophosphate - G_i, G_S Guanine nucleotide-binding proteins linked to inhibition and stimulation, respectively, of adenylate cyclase - GTP Guanosine-5-triphosphate - PDE Cyclic Nucleotide Phosphodiesterase - PGE₁ Prostaglandin E₁     

An improved purification procedure and properties of casein kinase II from brain

A simple and short purification procedure applicable to casein kinase II has been developed, for fully characterizing the enzyme from calf cerebral cortex cytosol. The procedure consists of four chromatographic steps: DEAE-cellulose, phosphocellulose, phosvitin-Sepharose and ATP-agarose which yields 87% pure casein kinase II. The purified enzyme shows three major bands with apparent molecular masses of 42, 38, and 27 kDa by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and is self-autophosphorylated on its 27 kDa polypeptide. The enzyme shows all the characteristics described for casein kinase II from other sources: it is independent of cyclic nucleotides, calcium/phospholipids, and double-stranded poly(I).poly(C); it can utilize both ATP and GTP as phosphoryl donors and can phosphorylate both casein and phosvitin but not histone. The kinetic studies establish that theK _m for ATP is 12.5 M and 25.1 M when using phosvitin and casein respectively as phosphoryl acceptors. TheK _m for phosvitin is 0.91 mg/ml and for casein 1.43 mg/ml, while theV _max is 315 nmol/min/per mg protein and 479 nmol/min/per mg protein for phosvitin and casein respectively. The activity of the kinase is highly stimulated by KCl or NaCl, and almost completely inhibited by heparin concentrations of 1 g/ml (92%). This inhibition is reduced to only 33% in the presence of optimal KCl concentrations (150 mM). Spermine stimulates enzyme activity, whilst hemin produces a slight inhibition.