Phosphorylation of glycogen synthase I from human polymorphonuclear leukocytes

Summary Glycogen synthase I from human polymorphonuclear leukocytes was phosphorylated with cAMP dependent protein kinase, synthase kinase or phosvitin kinase prepared from these cells. Limited tryptic hydrolysis released four phosphopeptides (t-A, t-B, t-C, t-D). Subsequent α-chymotryptic hydrolysis of the trypsin resistant core released three phosphopeptides. (c-A, c-B, c-C). The kinetic changes of glycogen synthase were compared with the phosphorylation of the peptides. Equivalent kinetic changes (K_c=0.2–0.3 mM Glc-6-P) were obtained when 1 P_i/subunit was introduced by cAMP dependent protein kinase, 0.5 P_i/subunit by synthase kinase and 0.8 P_i/subunit by both kinases. Initially, cAMP dependent protein kinase phosphorylated peptides c-A and t-C in parallel and somewhat later also t-B, whereas synthase kinase initially phosphorylated only c-A. The ultimate effect of the two kinases on c-A was additive. It was concluded that the initial kinetic changes were dependent on phosphorylation of c-A, which contained two sites, one for each kinase. The same kinetic changes were induced by phosphorylation on each of the sites. In the subsequent phosphorylation the kinases, separately or together, phosphorylated peptide c-C indicating one non-specific phosphorylatable site in this peptide. The cAMP dependent protein kinase alone phosphorylated t-C maximally, whereas both kinases were required for an equal phosphorylation of t-A and t-B. It is suggested that the cAMP dependent protein kinase phosphorylated t-A and t-C, whereas the data did not allow a similar suggestion for t-B. The kinetic changes occurring during the later stages of phosphorylation were an increase in K_c for Glc. 6-P to 4–5 mM at 1.85 P_i/subunit and to 20 mM at 3.3 P_i/subunit, but the changes could not be assigned to phosphorylation of any specific peptide. Phosphorylation of the peptides t-D and c-B were insignificant, but c-B may be phosphorylated under other experimental conditions (25). The phosvitin kinase phosphorylated glycogen synthase extremely slowly to an extent of 0.8 P_i/subunit, mainly in peptide c-C. Glycogen synthase would appear without physiological importance as substrate for this kinase. Phosphorylase kinase from rabbit skeletal muscle incorporated 0.7 P_i/subunit, mainly in peptide c-A causing a decrease in RI to 0.3, which upon further incubation remained constant. The rate of decrease in RI to 0.5 was unaffected by several synthase modifiers, including Glc-6-P, but was inhibited by ADP and P_i. The rate of phosphorylation by cAMP dependent protein kinase and synthase kinase was diversely affected in different buffers, however, without affecting the ultimate phosphorylation pattern.     

Phosphorylation of rabbit skeletal muscle glycogen synthase 1 by the cAMP dependent protein kinase,the cAMP independent synthase kinase and the phosvitin kinase from human polymorphonuclear leukocytes

Summary cAMP dependent protein kinase and cAMP independent synthase kinase incorporated up to two P_i/subunit in rabbit skeletal muscle glycogen synthase I. The first P_i/subunit was incorporated much faster than the second. After incorporation of one Pi/subunit by the CAMP dependent protein kinase, the ratio of independence (RI) was 0.20 and the dissociation constant K_c for Glc-6-P was 0.3 mm, and quite different from the RI of 0.02 and K_c (Glc-6-P) of 1 mM, obtained when one P_i/subunit was incorporated by the cAMP independent synthase kinase. Within the first P_i/subunit, the cAMP dependent protein kinase predominantly phosphorylated in the trypsin sensitive region (60–70%), corresponding to two trichloro-acetic acid soluble tryptic phosphopeptides, termed site-1 and site-2. Site-2 was found to be phosphorylated prior to site-1. CNBr degradation resolved the phosphorylated regions in two phosphopeptides with M_r 28,000 and 10,000.The larger CNBr phosphopeptides were derived from the trypsin sensitive region. Within the first P_i/subunit, synthase kinase almost exclusively phosphorylated in the trypsin insensitive region (80%) corresponding to the smaller CNBr phosphopeptide. However, when two P_i/subunit were incorporated by either the cAMP dependent protein kinase or the synthase kinase the phosphates were almost equally distributed between the trypsin sensitive and insensitive regions and K_c (Glc-6-P) increased to 2 mm, Maximum phosphorylation (2.8–3.3 P_i/subunit and K_c (Glc-6-P) 9–11 mm) was only obtainable when both the cAMP dependent protein kinase and the synthase kinase were present.The phosvitin kinase very slowly incorporated one P_i/subunit.We suggest that within the first P₁subunit phosphorylation in the trypsin insensitive region determine the affinity for the allosteric activator, glucose-6-phosphate. Thereafter phosphorylation in the trypsin sensitive region is the major determinant. Purified glycogen-free rabbit skeletal muscle glycogen synthase binds glycogen with lower affinity than polymorphonuclear leukocyte glycogen synthase. Glycogen was found to increase the initial rate of phosphorylation and facilitate the phosphorylation of site-1.Abbreviations cAMP adenosine cyclic 3:5-monophosphate - Glc-6-P glucose-6-phosphate - UDP-Glc uridine 5-diphosphoglucose - EGTA ethylene glycol-bis(-aminoethylether)-N,N-tetraacetic acid - EDTA ethylenediamine tetraacetic acid - CNBr cyanogen bromide - DTT dithiothreitol - SDS sodium dodecyl sulphate - RI ratio of independence     

Reversible inhibition of skeletal muscle phosphoprotein phosphatase by ATP, phosphate and fluoride.

A phosphoprotein phosphatase preparation which showed activity towards glycogen synthase, phosphorylase, phosphorylase kinase, and phosphohistones was reversibly inhibited (70–90%) by preincubation with free ATP (apparent K_i about 0.3 mM). Other nucleotides (ADP2 (apparent K_a 3μM) prior to assay. Other divalent metals (Co⁺⁺ > Zn⁺⁺ > Mg⁺⁺) were partially effective in reversing the inhibition. It is concluded that ATP by virtue of its special structure and metal binding capacity possibly removes a catalytically important metal ion from the enzyme.     

Purification and characterization of trehalose phosphorylase from the commercial mushroom Agaricus bisporus

Trehalose phosphorylase (EC 2.4.1.64) from Agaricus bisporus was purified for the first time from a fungus. This enzyme appears to play a key role in trehalose metabolism in A. bisporus since no trehalase or trehalose synthase activities could be detected in this fungus. Trehalose phosphorylase catalyzes the reversible reaction of degradation (phosphorolysis) and synthesis of trehalose. The native enzyme has a molecular weight of 240 kDa and consists of four identical 61-kDa subunits. The isoelectric point of the enzyme was pH 4.8. The optimum temperature for both enzyme reactions was 30°C. The optimum pH ranges for trehalose degradation and synthesis were 6.0–7.5 and 6.0–7.0, respectively. Trehalose degradation was inhibited by ATP and trehalose analogs, whereas the synthetic activity was inhibited by P_i (K_i=2.0 mM). The enzyme was highly specific towards trehalose, P_i, glucose and α-glucose-1-phosphate. The stoichiometry of the reaction between trehalose, P_i, glucose and α-glucose-1-phosphate was 1:1:1:1 (molar ratio). The K_m values were 61, 4.7, 24 and 6.3 mM for trehalose, P_i, glucose and α-glucose-1-phosphate, respectively. Under physiological conditions, A. bisporus trehalose phosphorylase probably performs both synthesis and degradation of trehalose.     

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

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

Phosphorylation and inactivation of liver glycogen synthase by liver protein kinases

A rapid method for purifying glycogen synthase a from rat liver was developed and the enzyme was tested as a substrate for nine different protein kinases, six of which were isolated from rat liver. The enzyme was phosphorylated on a 17-kDa CNBr fragment to approximately 1 phosphate/87-kDa subunit by phosphorylase b kinase from muscle or liver with a decrease in the activity ratio (-Glc-6-P/+Glc-6-P) from 0.95 to 0.6. Calmodulin-dependent glycogen synthase kinase from rabbit liver produced a similar phosphorylation pattern, but a smaller activity change. The catalytic subunit of beef heart cAMP-dependent protein kinase incorporated greater than 1 phosphate/subunit initially into a 17-kDa CNBr peptide and then into a 27-30-kDa CNBr peptide, with an activity ratio decrease to 0.5. Glycogen synthase kinases 3, 4, and 5 and casein kinase 1 were purified from rat liver. Glycogen synthase kinase 3 rapidly phosphorylated liver glycogen synthase to 1.5 phosphate/subunit with incorporation of phosphate into 3 CNBr peptides and a decrease in the activity ratio to 0.3. Glycogen synthase kinase 4 produced a pattern of phosphorylation and inactivation of liver synthase which was very similar to that caused by phosphorylase b kinase. Glycogen synthase kinase 5 incorporated 1 phosphate/subunit into a 24-kDa CNBr peptide, but did not alter the activity of the synthase. Casein kinase 1 phosphorylated and inactivated liver synthase with incorporation of phosphate into a 24-kDa CNBr peptide. This kinase and glycogen synthase kinase 4 were more active against muscle glycogen synthase. Calcium-phospholipid-dependent protein kinase from brain phosphorylated liver and muscle glycogen synthase on 17- and 27-kDa CNBr peptides, respectively. However, there was no change in the activity ratio of either enzyme. The following conclusions are drawn. 1) Liver glycogen synthase a is subject to multiple site phosphorylation. 2) Phosphorylation of some sites does not per se control activity of the enzyme under the assay conditions used. 3) Liver contains most, if not all, of the protein kinases active on glycogen synthase previously identified in skeletal muscle.     

Characterization of GSK-M, a glycogen synthase kinase from rat skeletal muscle

A form of glycogen synthase kinase designated GSK-M3 was purified 4000-fold from rat skeletal muscle by phosphocellulose, Affi-Gel blue, Sephacryl S-300 and carboxymethyl-Sephadex column chromatography. Separation of GSK-M from the catalytic subunit of the cAMP-dependent protein kinase was facilitated by converting the catalytic subunit to the holoenzyme form by addition of the regulatory subunit prior to the gel filtration step. GSK-M had an apparent Mr 62,000 (based on gel filtration), an apparent Km of 11 microM for ATP, and an apparent Km of 4 microM for rat skeletal muscle glycogen synthase. The kinase had very little activity with 0.2 mM GTP as the phosphate donor. Kinase activity was not affected by the addition of cyclic nucleotides, EGTA, heparin, glucose 6-P, glycogen, or the heat-stable inhibitor of cAMP-dependent protein kinase. Phosphorylation of glycogen synthase from rat skeletal muscle by GSK-M reduced the activity ratio (activity in the absence of Glc-6-P/activity in the presence of Glc-6-P X 100) from 90 to 25% when approximately 1.2 mol of phosphate was incorporated per mole of glycogen synthase subunit. Phosphopeptide maps of glycogen synthase obtained after digestion with CNBr or trypsin showed that this kinase phosphorylated glycogen synthase in serine residues found in the peptides containing the sites known as site 2, which is located in the N-terminal CNBr peptide, and site 3, which is located in the C-terminal CNBr peptide of glycogen synthase. In addition to phosphorylating glycogen synthase, GSK-M phosphorylated inhibitor 2 and activated ATP-Mg-dependent protein phosphatase. Activation of the protein phosphatase by GSK-M was dependent on ATP and was virtually absent when ATP was replaced with GTP. GSK-M had minimal activity toward phosphorylase b, casein, phosvitin, and mixed histones. These data indicate that GSK-M, a major form of glycogen synthase kinase from rat skeletal muscle, differs from the known glycogen synthase kinases isolated from rabbit skeletal muscle.     

Phosphorylation of rat liver glycogen synthase by phosphorylase kinase

Phosphorylation of rat liver glycogen synthase by rabbit skeletal muscle phosphorylase kinase results in the incorporation of approximately 0.8-1.2 mol of PO4/subunit. Analyses of the tryptic peptides by isoelectric focusing and thin layer chromatography reveal the presence of two major 32P-labeled peptides. Similar results were obtained when the synthase was phosphorylated by rat liver phosphorylase kinase. This extent of phosphorylation does not result in a significant change in the synthase activity ratio. In contrast, rabbit muscle glycogen synthase is readily inactivated by rabbit muscle phosphorylase kinase; this inactivation is further augmented by the addition of rabbit muscle cAMP-dependent protein kinase or cAMP-independent synthase (casein) kinase-1. Addition of cAMP-dependent protein kinase after initial phosphorylation of liver synthase with phosphorylase kinase, however, does not result in an inactivation or additional phosphorylation. The lack of additive phosphorylation under this condition appears to result from the phosphorylation of a common site by these two kinases. Partial inactivation of liver synthase can be achieved by sequential phosphorylation with phosphorylase kinase followed by synthase (casein) kinase-1. Under this assay condition, the phosphate incorporation into the synthase is additively increased and the synthase activity ratio (-glucose-6-P/+glucose-6-P) is reduced from 0.95 to 0.6. Nevertheless, if the order of the addition of these two kinases is reversed, neither additive phosphorylation nor inactivation of the synthase is observed. Prior phosphorylation of the synthase by phosphorylase kinase transforms the synthase such that it becomes a better substrate for synthase (casein) kinase-1 as evidenced by a 2- to 4-fold increase in the rate of phosphorylation. This increased rate of phosphorylation of the synthase appears to result from the rapid phosphorylation of a site neighboring that previously phosphorylated by phosphorylase kinase.     

The phosphorylation of rabbit skeletal muscle glycogen synthase by glycogen synthase kinase-2 and adenosine-3':5'-monophosphate-dependent protein kinase.

Purified glycogen synthase is contaminated with traces of two protein kinases that can phosphorylate the enzyme. One is protein kinase dependent on adenosine 3':5'-monophosphate (cyclic AMP) and the second is an activity termed glycogen synthase kinase-2 [Nimmo, H.G. and Cohen P, (1974)]. Glycogen synthase kinase-2 has been found to be localized relatively specifically in the protein-glycogen complex. It has been purified 4000-fold by two procedures, both of which involve disruption of the complex, followed by the DEAE-cellulose and phosphocellulose chromatographies. However the salt concentration at which glycogen synthase kinase-2 is eluted from DEAE-cellulose depends on the method that is used to disrupt the complex. The results indicate that glycogen synthase kinase-2 is firmly attached to a protein component of the complex. The isolation procedures separate glycogen synthase kinase-2 from phosphorylase kinase, cyclic AMP-dependent protein kinase and other glycogen-metabolising enzymes. Glycogen synthase kinase-2 is the major phosvitin kinase in skeletal muscle, although glycogen synthase is a six to eight-fold better substrate than phosvitin under the standard assay conditions. Phosphorylase kinase and phosphorylase b are not substrates for glycogen synthase kinase 2. Following incubation with cyclic-AMP-dependent protein kinase, cyclic AMP and Mg-ATP, the phosphorylation of glycogen synthase reaches a plateau at 1.0 molecules of phosphate incorporated per subunit and the activity ratio measured in the absence and presence of glucose 6-phosphate falls from 0.8 to a plateau of 0.18. The Ka for glucose 6-phosphate of this phosphorylated species, termed glycogen synthase b1, is the 0.6 mM. Following incubation with glycogen synthase kinase-2 and Mg-ATP, the phosphorylation reaches a plateau of 0.92 molecules of phosphate incorporated per subunit and the activity ratio decreases to a plateau of 0.08. The Ka for glucose 6-phosphate of this phosphorylated species, termed glycogen synthetase b2, is 4 mM. In the presence of both cyclic-AMP-dependent protein kinase and glycogen synthase kinase-2, the phosphorylation of glycogen synthase reaches a plateau when 1.95 molecules of phoshophate have been incorporated per subunit. The activity ratio is 0.01 and the Ka for glucose 6-phosphate is 10 mM. The results indicate that glycogen synthase can be regulated by two distinct phosphorylation-dephosphorylation cycles. The implication of these findings for the regulation of glycogen synthase in vivo are discussed.     

Protein phosphatase-1 and insulin action

Protein Phosphatase-1 (PP-1) appears to be the key component of the insulin signalling pathway which is responsible for bridging the initial insulin-simulated phosphorylation cascade with the ultimate dephosphorylation of insulin sensitive substrates. Dephosphorylations catalyzed by PP-1 activate glycogen synthase (GS) and simultaneously inactivate phosphorylase a and phosphorylase kinase promoting glycogen synthesis. Our in vivo studies using L6 rat skeletal muscle cells and freshly isolated adipocytes indicate that insulin stimulates PP-1 by increasing the phosphorylation status of its regulatory subunit (PP-1_G). PP-1 activation is accompanied by an inactivation of Protein Phosphatase-2A (PP-2A) activity. To gain insight into the upstream kinases that mediate insulin-stimulated PP-1_G phosphorylation, we employed inhibitors of the ras/MAPK, PI3-kinase, and PKC signalling pathways. These inhibitor studies suggest that PP-1_G phosphorylation is mediated via a complex, cell type specific mechanism involving PI3-kinase/PKC/PKB and/or the ras/MAP kinase/Rsk kinase cascade. cAMP agonists such as SpcAMP (via PKA) and TNF- (recently identified as endogenous inhibitor of insulin action via ceramide) block insulin-stimulated PP-1_G phosphorylation with a parallel decrease of PP-1 activity, presumably due to the dissociation of the PP-1 catalytic subunit from the regulatory G-subunit. It appears that any agent or condition which interferes with the insulin-induced phosphorylation and activation of PP-1, will decrease the magnitude of insulin's effect on downstream metabolic processes. Therefore, regulation of the PP-1_G subunit by site-specific phosphorylation plays an important role in insulin signal transduction in target cells. Mechanistic and functional studies with cell lines expressing PP-1_G subunit site-specific mutations will help clarify the exact role and regulation of PP-1_G site-specific phosphorylations on PP-1 catalytic function.     

Action of 5-thio-D-glucose in the control of glycogen depolymerization

With muscle glycogen phosphorylase a and b, 5-thio-$\text{D}$-glucose is a non-competitive inhibitor toward phosphate where it has a K_i of 13 mM and 5.1 mM, respectively, and produces a mixed type of inhibition when glycogen is the substrate.5-Thio-$\text{D}$-glucose enhances diaphragm phosphorylase phosphatase activity to the same extent as $\text{D}$-glucose, yet the thioanalog does not affect phosphorylase b kinase. Thus, the action of 5-thio-$\text{D}$-glucose on glycogen degradation proceeds by inhibition of phosphorylase a and b and by inactivation of phosphorylase a through converting it to the b form.     

Ligand-induced conformations of rat brain hexokinase: studies with hexoses, hexose 6-phosphates, and nucleoside triphosphates leading to development of a new model for the enzyme

Various ligands of rat brain hexokinase (ATP:d-hexose 6-phosphotransferase, EC 2.7.1.1) have been found to protect the enzyme against either (or both) chymotryptic digestion or inactivation by glutaraldehyde. Using this protective effect, the K_d for various enzyme-ligand complexes has been estimated: hexokinase-Glc, K_d = 0.24 ± 0.03mM (chymotryptic digestion), K_d = 0.26 ± 0.07mM (glutaraldehyde inactivation); hexokinase-Glc-6- P, K_d = 0.041 ± 0.005m M (glutaraldehyde inactivation); hexokinase-ATP, K_d = 1.01 ± 0.28mM (chymotryptic digestion); hexokinase-ATP-Mg ²⁺, K_d = 0.07-0.08mM (chymotryptic digestion). Other nucleoside triphosphates (UTP, ITP, GTP, and CTP) were much less effective than ATP at protecting against chymotrypsin. Various hexoses were tested for their ability to protect against glutaraldehyde. Only ?good” substrates (mannose, 2-deoxyglucose) protected; nonsubstrates (galactose, arabinose) and N-acetylglucosamine, a competitive inhibitor of Glc binding, were not effective. Various hexose 6-phosphates were tested for their ability to protect against glutaraldehyde inactivation. Glc-6-P was much more effective than were mannose-6-P, galactose-6-P, or fructose-6-P. It was observed that ?good” substrates (Glc, mannose) increased the effectiveness of Glc-6-P at solubilizing the mitochondrial form of the enzyme; galactose and N-acetylglucosamine had no effect on solubilization by Glc-6-P. These results are taken as an indication of enhanced Glc-6-P binding in the presence of Glc, as previously reported by Ellison et al. (J. Biol. Chem., 250, 1864–1871, 1975). Along with previous studies on ligand-induced conformations and kinetics of this enzyme, these results form the basis for a new model for brain hexokinase. This model specifically takes into account the ligand-induced conformations at various points in the catalytic cycle and specifically accounts for the ability of various hexoses to serve as substrates and hexose 6-phosphates to serve as inhibitors in terms of their ability to induce specific conformations of the enzyme. The properties of the various conformations involved in the model are designated by a four-letter code which facilitates comparison and discussion.     

Proton relay system in the active site of maltodextrinphosphorylase via hydrogen bonds with large proton polarizability: an FT-IR difference spectroscopy study

Maltodextrinphosphorylase (MDP) was studied in the pH range 5.4–8.4 by Fourier transform infrared (FT-IR) spectroscopy. The pK _a value of the cofactor pyridoxalphosphate (PLP) was found between 6.5 and 7.0, which closely resembles the second pK _a of free PLP. FT-IR difference spectra of the binary complex of MDP+α-d-glucose-1-methylenephosphonate (Glc-1-MeP) minus native MDP were taken at pH 6.9. Following binary complex formation, two Lys residues, tentatively assigned to the active site residues Lys533 and Lys539, became deprotonated, and PLP as well as a carboxyl group, most likely of Glu637, protonated. A system of hydrogen bonds which shows large proton polarizability due to collective proton tunneling was observed connecting Lys533, PLP, and Glc-1-MeP. A comparison with model systems shows, furthermore, that this hydrogen bonded chain is highly sensitive to local electrical fields and specific interactions, respectively. In the binary complex the proton limiting structure with by far the highest probability is the one in which Glc-1-MeP is singly protonated. In a second hydrogen bonded chain the proton of Lys539 is shifted to Glu637. In the binary complex the proton remains located at Glu637. In the ternary complex composed of phosphorylase, glucose-1-phosphate (Glc-1-P), and the nonreducing end of a polysaccharide chain (primer), a second proton may be shifted to the phosphate group of Glc-1-P. In the doubly protonated phosphate group the loss of mesomeric stabilization of the phosphate ester makes the C₁–O₁ bond of Glc-1-P susceptible to bond cleavage. The arising glucosyl carbonium ion will be a substrate for nucleophilic attack by the nonreducing terminal glucose residue of the polysaccharide chain. Received: 15 June 1997 / Revised version: 15 October 1998 / Accepted: 15 October 1998     

Acid phosphatases excreted by Humicola lutea 120-5 in casein-containing medium

The fungus Humicola lutea 120-5 cultivated in casein-containing media, in the presence or absence of inorganic phosphate (P_i), excretes three different molecular forms of acid phosphatase (with M_r values of approximately 140, 70 and 35 kDa). The enzyme forms were isolated and purified 30–100-fold by a procedure involving two steps of ion-exchange chromatography and Sephadex G-200 gel chromatography. It was found that the fungus excretes only one of the phosphatases with the highest M_r (140 kDa) during growth on medium with inorganic nitrogen source (NaNO₃). This form (designed AcPh I) was assumed to be a constitutive, since it showed resistance to high P_i-concentrations (10 mM) and its biosynthesis was not affected by the type of nitrogen source (casein or NaNO₃). The other two forms (AcPh II-70 and AcPh III-35 kDa) were competitively inhibited by P_i (K _i = 0.5 and 0.2 mM, respectively) and were induced by casein. The K _m values of AcPh I and AcPh II were estimated as 1.3 mM, while AcPh III showed a higher affinity for p-nitrophenylphosphate (pNPP) with K _m of 0.5 mM. The AcPh I–III fractions demonstrated a pH optimum in the range of 4.5–4.8 and an optimal temperature of 55 °C using pNPP as a substrate. This revised version was published online in November 2006 with corrections to the Cover Date.     

Human serum albumin coordinates Cu(II) at its N-terminal binding site with 1 pM affinity

The conditional stability constant at pH 7.4 for Cu(II) binding at the N-terminal site (NTS) of human serum albumin (HSA) was determined directly by competitive UV–vis spectroscopy titrations using nitrilotriacetic acid (NTA) as the competitor in 100 mM NaCl and 100 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (Hepes). The log K _NTS^c value of 12.0 ± 0.1 was determined for HSA dissolved in 100 mM NaCl. A false log log K _NTS^c value of 11.4 ± 0.1 was obtained in the 100 mM Hepes buffer, owing to the formation of a ternary Cu(NTA)(Hepes) complex. The impact of the picomolar affinity of HSA for Cu(II) on the availability of these ions in neurodegenerative disorders is briefly discussed.     

6-Phosphofructo-2-kinase and D-fructose-2,6-bisphosphatase activities in mung bean seedlings

6-Phosphofructo-2-kinase (ATP: D-fructose-6-phosphate-2-phosphotransferase) and D-fructose-2,6-bisphosphatase activities have been found in extracts prepared from etiolated mung bean seedlings. The activity of 6-phosphofructo-2-kinase exhibits a sigmoidal shape in response to changes in concentrations of both substrates, D-fructose 6-phosphate and ATP (S_0.5 values of 1.8 and 1.2 mM, respectively). Inorganic orthophosphate (P_i) has a strong stimulating effect on the 2-kinase activity (A_0.5 at about 2 mM), moderately increasing the V_max and modifying the response into hyperbolic curves with K_m values of 0.4 and 0.2 mM for fructose 6-phosphate and ATP, respectively. 3-Phosphoglycerate (I_0.5 about 0.15 mM) partially inhibited the kinase activity by counteracting the P_i activation. In contrast, the activity of D-fructose-2,6-bisphosphatase (K_m 0.38 mM) is strongly inhibited by P_i (I_0.5 0.8 mM) lowering its affinity to fructose-2,6-P₂ (K_m 1.4 mM). 3-Phosphoglycerate activites the enzyme (A_0.5 at about 0.3 mM) without causing a significant change in its K_m for fructose-2,6-P₂. The activities of both of these enzymes in relationship to the metabolic role of D-fructose 2,6-bisphosphate in the germinating seed is discussed.     

Glycogen synthase: the existence of a single demonstrable from in bovine adipose tissue.

Glycogen synthase from bovine adipose tissue has been kinetically characterized. Glucose 6-phosphate increased enzyme activity 50-fold with an activation constant (A_0.5) of 2.6 mm. Mg²⁺ reversibly decreased this A_0.5 to 0.75 mm without changing the amount of stimulation by glucose 6-phosphate. Mg²⁺ did not alter the apparent K_m for UDP-glucose (0.13 mm). The pH optimum was broad and centered at pH 7.6. The glucose 6-phosphate activation of the enzyme was reversible and competitively inhibited by ATP (K_i = 0.6 mm) and P_i(K_i = 2.0 mm). The use of exogenous sources of glycogen synthase and glycogen synthase phosphatase suggests that (i) adipose tissue glycogen synthase phosphatase activity in fed mature steers is low or undetectable, and (ii) endogenous bovine adipose tissue glycogen synthase can be activated to other glucose 6-phosphate-dependent forms by addition of adipose tissue extracts from fasted steers or fed rats.     

Syntheses of 1-[2-(Phosphonomethoxy)Alkyl]Thymine Monophosphates and an Evaluation of their Inhibitory Activity Toward Human Thymidine Phosphorylase

A series of new monophosphates of 1-[2-(phosphonomethoxy)alkyl]thymines, such as PMPTp_, 3-MeO-PMPTp, HPMPTp, and FPMPTp, were synthesized and tested for their ability to inhibit human thymidine phosphorylase. Kinetic measurements of enzyme activity were performed using thymidine and inorganic phosphate as the substrates. The data show that some monophosphates provide a considerable increase of the multisubstrate inhibitory effect. The highest inhibitory potency was found with (R)-FPMPTp 4c (K _i ^dT = 4.09 ± 0.47 μM, K _i(P_i) = 2.13 ± 0.29 μM) and (R) 3-MeO-PMPTp 4d (K _i ^dT = 5.78 ± 0.71 μM, K _i(P_i) = 2.71 ± 0.37 μM).     

Inactivation of phosphofructokinase 2 by cyclic AMP - dependent protein kinase

Treatment with the catalytic subunit of cyclic AMP-dependent protein kinase induced the following modifications in the kinetic properties of purified phosphofructokinase 2. The affinity for Fru-6-P, the V_max and the stimulatory effect of P_i were decreased; the inhibitory actions of P-enol-pyruvate and citrate were increased; the pH activity curve, measured in the presence of 5 mM Fru-6-P and 5 mM P_i was modified in the respect that the peak of activity normally measured at pH 6.6 was abolished whereas no effect of the treatment was observed at pH 8. Similar changes in the properties of phosphofructokinase 2 were also observed in a crude preparation obtained from hepatocytes incubated with glucagon.     

Reductive transformation of TNT by Escherichia coli resting cells: kinetic analysis

Microbial 2,4,6-trinitrotoluene (TNT) biotransformation via sequential nitro-reduction appears a ubiquitous process, but the kinetics of these transformations have been poorly understood or described. TNT transformation by Escherichia coli was monitored and a kinetic model for reductive TNT depletion was developed and experimentally calibrated in this report. Using resting cells of aerobically pregrown E. coli, TNT was quickly reduced to hydroxylaminodinitrotoluenes. The standard Michaelis–Menten model was modified to include three additional parameters: product toxicity (T _c), substrate inhibition (K _i), and intracellular reducing power (RH) limitation. Experimentally measured product toxicity (5.2 μmol TNT/mg cellular protein) closely matched the best-fit model value (2.84 μmol TNT/mg cellular protein). Parameter identifiability and reliability (k _m, K _s, T _c, and K _i) was evaluated and confirmed through sensitivity analyses and via Monte Carlo simulations. The resulting kinetic model adequately described TNT reduction kinetics by E. coli resting cells in the absence or presence of reducing power limitation.