Freezing tolerance/intolerance and cryoprotectant synthesis in terrestrially overwintering anurans in the Great Plains,USA

Mechanistic bases for freezing tolerance in anurans have been well-studied only in wood frogs, Rana sylvatica, so comprehensive explanations for the mechanisms and evolution of freezing tolerance in anurans are lacking. We measured crystallization temperatures, freezing tolerance/intolerance, and tissue glucose and glycogen phosphorylase activities in frozen and unfrozen winter-acclimated Pseudacris triseriata, Bufo cognatus and B. woodhousei. Freezing occurred at higher subzero temperatures on wet substrate than on dry substrate in all species, indicating susceptibility to inoculative freezing. P. triseriata was freeze-tolerant, but survival was dependent on the level of supercooling prior to freezing. All Bufo were freezing intolerant, regardless of crystallization temperature. Glucose was significantly elevated by freezing in both liver (35-fold) and leg muscle (22-fold) in winter P. triseriata, but only liver glucose was significantly elevated in B. cognatus. However, freezing did not alter glycogen phosphorylase activity in either species. Liver phosphorylase activity was significantly higher in P. triseriata than in B. cognatus, suggesting that capacity for mobilizing glucose from liver glycogen is associated with freezing tolerance. Summer measurements of liver phosphorylase activity, however, did not differ between species. Thus, P. triseriata, but not B. cognatus, exhibited winter increment of liver phosphorylase activity that is correlated with the development of freezing tolerance.Abbreviation T _b body temperature - T _c crystallization temperature - T _r rebound temperature - T _eq equilibrium temperature     

Regulation of the deo operon in Escherichia coli: the double negative control of the deo operon by the cytR and deoR repressors in a DNA directed in vitro system.

Summary The synthesis of the four enzymes of the deo operon in Escherichia coli is known from in vivo experiments to be subject to a double negative control, exerted by the products of the cytR and deoR genes.A DNA-directed in vitro protein synthesizing system makes the deo enzymes (exemplified by thymidine phosphorylase) in agreement with in vivo results. Enzyme synthesis is stimulated by cyclic AMP and repressed by the cytR and deoR gene products. Repression by the cytR repressor is reversed by cytidine or adenosine in the presence of cyclic AMP, while repression by the deoR repressor is reversed by deoxyribose-5-phosphate.Assays for the presence of the cytR and deoR repressors were established by use of S-30 extracts prepared from the regulatory mutants.Dissociation constants for repressor-operator binding as well as for repressor-inducer interactions have been estimated from the results.Abbreviations and Symbols deoA (previously designated tpp) Genes coding for: thymidine, phosphorylase - deoB (previously designated drm) deoxyribomutase - deoC (previously designated dra) deoxyriboaldolase - deoD (previously designated pup) purine nucleoside phosphorylase - udp uridine phosphorylase - cytR regulatory gene for cdd, udp, deoC, deoA, deoB, and deoD - deoR (previously designated nucR) regulatory gene for deoC, deoA, deoB, and deoD Enzymes (EC 2.4.2.1) Purine nucleoside phosphorylase or purine nucleoside: orthophosphate(deoxy)ribosyltansferase - (EC 2.4.2.4) thymidine phosphorylase or thymidine: orthophosphate deoxyribosyltransferase - (EC 2.4.2.3) uridine phosphorylase or uridine: orthophosphate ribosyltransferase - (EC 4.1.2.4) deoxyriboaldolase or 2-deoxy-D-ribose-5-phosphate: acetaldehydelyase - (EC 2.7.5.6) phosphodeoxyribomutase The deo operon is defined as the gene cluster consisting of deoC deoA deoB deoD. The deo enzymes are the four enzymes encoded by the four genes of the deo operon. cAMP: cyclic adenosine 3,5-monophosphate. CRP: cyclic AMP receptor protein. dRib-5P: deoxyribose-5-phosphate. THUR: 3,4,5,6-tetrahydrouridine; EDTA: ethylene-diamine-tetra-acetate.     

Multiple regulation of nucleoside catabolizing enzymes in Escherichia coli: effects of 3:5'' cyclic AMP and CRP protein.

Summary The regulation of the synthesis of nucleoside metabolizing enzymes has been studied in cya and crp mutant strains of Escherichia coli.The synthesis of the cyt-enzymes, cytidine deaminase and uridine phosphorylase regulated by the cytR gene product, is activated by the cAMP-CRP complex. On the other hand the synthesis of the deoenzymes: deoxyriboaldolase, thymidine phosphorylase, phosphodeoxyribomutase and purine nucleoside phosphorylase, appears to be increased if an active cAMP-CRP complex cannot be formed.It also seems that nucleosides serve as poor carbon sources for cya and crp mutants; this could not solely be explained by low levels of nucleoside metabolizing enzymes nor by a deficiency in nucleoside uptake. Addition of casamino acids stimulated the growth of cya and crp mutants, with nucleosides as carbon sources. When grown on glucose and casamino acids growth could be stimulated by adenine and hypoxanthine nucleosides; these results suggest an impaired nitrogen metabolism in cya and crp mutants.Abbreviations and Symbols cAMP cyclic adenosine 3:5-monophosphate - CRP cAMP receptor protein. Genes coding for: adenyl cyclase - cya cAMP receptor protein - crp cytidine deaminase - cdd uridine phosphorylase - udp thymidine phosphorylase - tpp purine nucleoside phosphorylase - pup; cytR regulatory gene for cdd, udp, dra, tpp, drm, and pup - deoR regulatory gene for dra, tpp, drm, and pup     

Enterococcus faecalis utilizes maltose by connecting two incompatible metabolic routes via a novel maltose 6′‐phosphate phosphatase (MapP)

Similar to Bacillus subtilis, Enterococcus faecalis transports and phosphorylates maltose via a phosphoenolpyruvate (PEP):maltose phosphotransferase system (PTS). The maltose‐specific PTS permease is encoded by the malT gene. However, E. faecalis lacks a malA gene encoding a 6‐phospho‐α‐glucosidase, which in B. subtilis hydrolyses maltose 6′‐P into glucose and glucose 6‐P. Instead, an operon encoding a maltose phosphorylase (MalP), a phosphoglucomutase and a mutarotase starts upstream from malT. MalP was suggested to split maltose 6‐P into glucose 1‐P and glucose 6‐P. However, purified MalP phosphorolyses maltose but not maltose 6′‐P. We discovered that the gene downstream from malT encodes a novel enzyme (MapP) that dephosphorylates maltose 6′‐P formed by the PTS. The resulting intracellular maltose is cleaved by MalP into glucose and glucose 1‐P. Slow uptake of maltose probably via a maltodextrin ABC transporter allows poor growth for the mapP but not the malP mutant. Synthesis of MapP in a B. subtilis mutant accumulating maltose 6′‐P restored growth on maltose. MapP catalyses the dephosphorylation of intracellular maltose 6′‐P, and the resulting maltose is converted by the B. subtilis maltose phosphorylase into glucose and glucose 1‐P. MapP therefore connects PTS‐mediated maltose uptake to maltose phosphorylase‐catalysed metabolism. Dephosphorylation assays with a wide variety of phospho‐substrates revealed that MapP preferably dephosphorylates disaccharides containing an O‐α‐glycosyl linkage.     

A second purine nucleoside phosphorylase in Escherichia coli K-12

Summary The presence of a second purine nucleoside phosphorylase in wild-type strains of E. coli K-12 after growth on xanthosine has been demonstrated. Like other purine nucleoside phosphorylases it is able to carry out both phosphorylosis and synthesis of purine deoxy- and ribonucleosides whilst pyrimidine nucleosides cannot act as substrates. In contrast to the well characterised purine nucleoside phosphorylase of E. coli K-12 (encoded by the deoD gene) this new enzyme could act on xanthosine and is hence called xanthosine phosphorylase. Studies of its substrate specificity showed that xanthosine phosphorylase, like the mammalian purine nucleoside phosphorylases, has no activity towards adenine and the corresponding nucleosides. Determinations of K _m and gel filtration behaviour was carried out on crude dialysed extracts. The presence of xanthosine phosphorylase enables E. coli to grow on xanthosine as carbon source. Xanthosine was the only compound found which induced xanthosine phosphorylase. No other known nucleoside catabolising enzyme was induced by xanthosine. The implications of non-linear induction kinetics of xanthosine phosphorylase is discussed.     

An operator constitutive mutant affecting the synthesis of two enzymes involved in the catabolism of nucleosides in Escherichia coli

Summary A new type of mutant of mutant of Escherichia coli that synthesises thymidine phosphorylase constitutively has been isolated and characterised. The mutation leading to constitutivity is located to the left of the gene specifying deoxyriboaldolase. The mutation is cis dominant in its effect on thymidine phosphorylase activity and therefore believed to be a mutation of the operator-constitutive type. The specific activity of purine nucleoside phosphorylase is not affected by the mutation indicating that the gene specifying this enzyme is located in a different operon from that containing the genes specifying thymidine phosphorylase and deoxyriboaldolase.     

Induction and repression of enzymes involved in exogenous purine compound utilization in Bacillus cereus

5′-Nucleotidase, adenosine phosphorylase, adenosine deaminase and purine nucleoside phosphorylase, four enzymes involved in the utilization of exogenous purine compounds in Bacillus cereus, were measured in extracts of this organism grown in different conditions. It was found that adenosine deaminase is inducible by addition of adenine derivatives to the growth medium, and purine nucleoside phosphorylase by metabolizable purine and pyrimidine ribonucleosides. Adenosine deaminase is repressed by inosine, while both enzymes are repressed by glucose. Evidence is presented at during growth of B. cereus in the presence of AMP, the concerted action of 5′-nucleotidase and adenosine phosphorylase, two constitutive enzymes, leads to formation of adenine, and thereby to induction of adenosine deaminase. The ionsine formed would then cause induction of the purine nucleoside phosphorylase and repression of the deaminase. Taken together with our previous findings showing that purine nucleoside phosphorylase of B cereus acts as a translocase of the ribose moiety of ionsine inside the cell (Mura, U., Sgarrella, F. and Ipata, P.L. (1978) J. Biol. Chem. 253, 7905–7909), our results provide a clear picture of the molecular events leading to the utilization of the sugar moiety of exogenous AMP, adenosine and inosine as an energy source.     

Mutants of Escherichia coli unable to metabolize cytidine: isolation and characterization

Summary Strains of Escherichia coli have been selected, which contain mutations in the udk gene, encoding uridine kinase. The gene has been located on the chromosome as cotransducible with the his gene and shown to be responsible for both uridine and cytidine kinase activities in the cell.An additional mutation in the cdd gene (encoding cytidine deaminase) has been introduced, thus rendering the cells unable to metabolize cytidine. In these mutants exogenously added cytidine acts as inducer of nucleoside catabolizing enzymes indicating that cytidine per se is the actual inducer.When the udk, cdd mutants are grown on minimal medium the enzyme levels are considerably higher than in wild type cells. Evidence is presented indicating that the high levels are due to intracellular accumulation of cytidine, which acts as endogenous inducer.Abbreviations and Symbols FU 5-fluorouracil - FUR 5-fluorouridine - FUdR 5-fluoro-2'deoxyuridine - FCR 5-fluorocytidine - FCdR 5-fluorodeoxycytidine - THUR 3, 4, 5, 6-tetrahydrouridine - UMP uridine monophosphate - CMP cytidine monophosphate - dUMP deoxyuridine monophosphate. Genes coding for: cytidine deaminase - edd uridine phosphorylase - udp thymidine phosphorylase - tpp purmnucleoside phosphorylase - pup uridine kinase (=cytidine kinase) - udk UMP-pyrophosphorylase - upp. CytR regulatory gene for cdd, udp, dra, tpp, drm and pup Enzymes EC 2.4.2.1 Purine nucleoside phosphorylase or purine nucleoside: orthophosphate (deoxy)-ribosyltransferase - EC 2.4.2.4 thymidine phosphorylase or thymidine: orthophosphate deoxyribosyltransferase - EC 2.4.2.3 uridine phosphorylase or uridine: orthophosphate ribosyltransferase - EC 3.5.4.5 cytidine deaminase or (deoxy)cytidine aminohydrolase - EC 4.1.2.4 deoxyriboaldolase or 2-deoxy-D-ribose-5-phosphate: acetaldehydelyase - EC 2.4.2.9 UMP-pyrophosphorylase or UMP: pyrophosphate phosphoribosyltransferase - EC 2.7.1.48 uridine kinase or ATP: uridine 5-phosphotransferase     

Nonsense mutations in the amylomaltase gene and other loci of Streptococcus pneumoniae

Summary Maltose-negative mutations in the amylomaltase gene of Streptococcus pneumoniae were examined for the presence of nonsense mutations. Out of 28 single-site mutants tested, 3 were shown to be suppressible by an amber suppressor previously found by Gasc et al. (1979). In the presence of the suppressor these mutants manifested 10–30% of wild type amylomaltase activity. In addition to the amylomaltase governed by malM, and the maltosaccharide phosphorylase governed by malP (which maps to the side of malM distal to the regulatory gene, malR), a new maltose-inducible protein, governed by another gene, malX, was observed in gel electrophoretic patterns. The malX gene maps on the side of malM proximal to the malR gene. The approximate molecular weights of the amylomaltase, phosphorylase and malX polypeptides are 62,000, 87,000 and 50,000, respectively. There appear to be no polar effects of the nonsense mutations in the malM gene on synthesis of the gene products of either malP or malX. In a search for nonsense mutants at other loci, one was found in the end gene, which governs the major endonuclease, a membrane enzyme. None were detected among 5 mismatch-repair defective hex mutants analyzed.     

Glycogen phosphorylase from flight muscle of the hawk moth,Manduca sexta: purification and properties of three interconvertible forms and the effect of flight on their interconversion

Glycogen phosphorylase (EC 2.4.1.1) of Manduca sexta flight muscle was separated into three distinct peaks of activity on diethylaminoethyl-Sephacel. The three fractions of phosphorylase activity were further purified by affinity chromatography on AMP-Sepharose and shown to have the same relative molecular mass (=178000) on polyacrylamide gradient gel electrophoresis under non-denaturating conditions and to produce subunits of molecular mass =92000 on SDS gelelectrophoresis. On the basis of their kinetic properties with respect to the activator AMP and the inhibitor caffeine, the three fractions of phosphorylase activity were assigned as follows: peak 1=phosphorylase b (unphosphorylated form), peak 3=phosphorylase a (phosphorylated form); peak 2 represented a phospho-dephospho hybrid in which only one subunit of the dimeric enzyme was phosphorylated. This hypothesis was corroborated as the various forms could be interconverted in vitro by either dephosphorylation by an endogenous protein phosphatase producing the b form, or by phosphorylation catalyzed by purified phosphorylase kinase from rabbit muscle producing phosphorylase ab and a. From muscle of resting moths more phosphorylase was isolated in the b form (41%) than in the forms ab (28%) and a (31%), respectively. This proportion was changed in favour of the fully phosphorylated a form after a brief interval of flight when 68% of the phosphorylase activity was represented by the a form and only 13% by the b form. Unlike the phosphorylated forms a and ab of phosphorylase, the b form had low affinities for the substrates and for the activator AMP, and was virtually inactive if near-physiological concentrations of substrates and effectors were employed in the assays. The results demonstrate that in Manduca flight muscle three forms of phosphorylase coexist and that their interconversion is a mechanism for regulating phosphorylase activity in vivo.Abbreviations DEAE diethylaminoethyl - EDTA ethylenediamine tetraacetate - EGTA ethyleneglycol-bis(-aminoethylether)N,N-tetra-acetic acid - M _r relative molecular mass - NMR nuclear magnetic resonance - PAGGE polyacrylamide gradient gel electrophoresis - P_i morganic phosphate - SDS sodium dodecylsulphate - TRIS tris(hydroxymethyl)-aminomethane - V _max maximum activity     

Physiological aggregation of maltodextrin phosphorylase from Pyrococcus furiosus and its application in a process of batch starch degradation to α-d-glucose-1-phosphate

Maltodextrin phosphorylase from Pyrococcus furiosus (PF1535) was fused with the cellulose-binding domain of Clostridium cellulovorans serving as an aggregation module. After molecular cloning of the corresponding gene fusion construct and controlled expression in Escherichia coli BL21, 83% of total maltodextrin phosphorylase activity (0.24 U/mg of dry cell weight) was displayed in active inclusion bodies. These active inclusion bodies were easily isolated by nonionic detergent treatment and directly used for maltodextrin conversion to α-d-glucose-1-phosphate in a repetitive batch mode. Only 10% of enzyme activity was lost after ten conversion cycles at optimum conditions.     

Dinucleotide repeat polymorphism within the PHKA1 gene at Xq12-q13

A polymorphic complex repeat including two (TG) _n stretches was identified in the intron following codon 26 of the human gene encoding the muscle isoform of the phosphorylase kinase subunit (PHKA1). It should be a useful marker for linkage analysis of families with heritable phosphorylase kinase deficiency and for gene mapping in the vicinity of the X inactivation center.     

Reciprocal regulation of glycogen phosphorylase and glycogen synthase by insulin involving phosphatidylinositol-3 kinase and protein phosphatase-1 in HepG2 cells

The effect of insulin on glycogen synthesis and key enzymes of glycogen metabolism, glycogen phosphorylase and glycogen synthase, was studied in HepG2 cells. Insulin stimulated glycogen synthesis 1.83-3.30 fold depending on insulin concentration in the medium. Insulin caused a maximum of 65% decrease in glycogen phosphorylase 'a' and 110% increase in glycogen synthase activities in 5 min. Although significant changes in enzyme activities were observed with as low as 0.5 nM insulin level, the maximum effects were observed with 100 nM insulin. There was a significant inverse correlation between activities of glycogen phosphorylase 'a' and glycogen synthase 'a' (R² = 0.66, p < 0.001). Addition of 30 mM glucose caused a decrease in phosphorylase 'a' activity in the absence of insulin and this effect was additive with insulin up to 10 nM concentration. The inactivation of phosphorylase 'a' by insulin was prevented by wortmannin and rapamycin but not by PD98059. The activation of glycogen synthase by insulin was prevented by wortmannin but not by PD98059 or rapamycin. In fact, PD98059 slightly stimulated glycogen synthase activation by insulin. Under these experimental conditions, insulin decreased glycogen synthase kinase-3 activity by 30-50% and activated more than 4-fold particulate protein phosphatase-1 activity and 1.9-fold protein kinase B activity; changes in all of these enzyme activities were abolished by wortmannin. The inactivation of GSK-3 and activation of PKB by insulin were associated with their phosphorylation and this was also reversed by wortmannin. The addition of protein phosphatase-1 inhibitors, okadaic acid and calyculin A, completely abolished the effects of insulin on both enzymes. These data suggest that stimulation of glycogen synthase by insulin in HepG2 cells is mediated through the PI-3 kinase pathway by activating PKB and PP-1G and inactivating GSK-3. On the other hand, inactivation of phosphorylase by insulin is mediated through the PI-3 kinase pathway involving a rapamycin-sensitive p70^s6k and PP-1G. These experiments demonstrate that insulin regulates glycogen phosphorylase and glycogen synthase through (i) a common signaling pathway at least up to PI-3 kinase and bifurcates downstream and (ii) that PP-1 activity is essential for the effect of insulin.     

Location of phosphorylase kinase (Phk) in the mouse X chromosome

The phosphorylase kinase deficiency (Phk) locus has been located in the mouse X chromosome, the order of genes being centromere-Bn-Phk-Ta-jp. Since the Phk locus of the mouse may be identical to the locus responsible for the X-linked phosphorylase kinase deficiency trait of man, and there may be a high degree of gene-order homology in the X chromosome of all mammals, the location of Phk in the mouse reported here may aid in locating the phosphorylase kinase gene on the X chromosome of man.This research was supported by grants AM 13359 (to F.H.) and AM 14461 (to D.L.C.) from the National Institute of Arthritis and Metabolic Diseases, and by an allocation (to E.M.E.) from NIH General Research Support Grant RR-05545 from the Division of Research Resources to The Jackson Laboratory. F.H. is a recipient of a Research Career Development Award (AM 46 421) of the National Institute of Arthritis and Metabolic Diseases.     

Liver phosphorylase kinase: characterization of two interconvertible forms and partial purification of phosphorylase kinase α

Summary The presence of two interconvertible forms of phosphorylase kinase has been confirmed in rat liver extracts. The pH optimum of the nonactivated form (PhK b) was lower than the pH optimum of the activated form (PhK ) as reported by others (2). In the absence of calcium the K_m of PhK for phosphorylase b was 53 + 10 U/ ml with a V_m of 17 = 1 U/gm of tissue. The K_m of PhK for phosphorylase b was 20 + 2 U/ml with a V_m of 65 U/gm. Calcium stimulated both forms of phosphorylase kinase(A_0.5 0.03 M). In the presence of 0.1 M calcium the Km for phosphorylase b of both forms of the enzyme was reduced. In addition, calcium increased the V_m of both forms, but the effect was greater for PhK b than for PhK . The K_m of both forms of phosphorylase kinase for ATP was 0.05 mM and was unaffected by calcium. All of these studies were done using liver phosphorylase b as substrate. Conditions for assaying PhK activity virtually independent of PhK b activity also are indicated. This will enable the monitoring of interconversion reactions in tissue extracts.Phosphorylase kinase a was purified to near homogeneity using DEAE-cellulose, Sepharose 4B gel filtration and ATP affinity chromatography. The molecular weight was approximately 1 × 106. The pII profile, calcium requirements and kinetic constants were the same as those for PhK a in the crude extract.     

The phosphorylation of troponin B by phosphorylase b kinase in skeletal muscle of mice carrying the phosphorylase b kinase deficiency gene

In skeletal muscle of animals with the phosphorylase $\text{b}$ kinase deficiency gene there is < 1% of the normal activity to convert phosphorylase $\text{b}$ to $\text{a}$ in the presence of Ca⁺⁺, Mg⁺⁺, and ATP (1). Correspondingly, there is < 1% of the normal activity to phosphorylate phosphorylase $\text{b}$. Nevertheless, under the same conditions, these extracts catalyze the phosphorylation of troponin at a rate 57% of normal. Phosphorylase $\text{b}$ converting activity can be sedimented from skeletal muscle of control mice by centrifugation. This fraction isolated from I strain skeletal muscle extracts phosphorylates troponin at a rate 29–39% of the control. EGTA¹ (15 mM) inhibits troponin phosphorylation by 50–60% in this fraction from both strains. The EGTA inhibition is reversed by 15 mM Ca⁺⁺. Thus the phosphorylase $\text{b}$ kinase in skeletal muscle of animals with the phosphorylase $\text{b}$ kinase deficiency gene can phosphorylate troponin B, although it shows little or no activity with phosphorylase as a substrate. This observation is consistent with the normal muscle contractility of I strain animals.     

Purification and characterization of glycogen phosphorylase A and B from the freeze-avoiding gall moth larvae Epiblema scudderiana

The active a and inactive b forms of glycogen phosphorylase from cold-hardy larvae of the gall moth, Epiblema scudderiana, were purified using DEAE⁺ ion exchange and 3-5-AMP-agarose affinity chromatography. Maximum activities for glycogen phosphorylases a and b were 6.3±0.74 and 2.7±0.87 mol glucose-1-P·min^-1·g wet weight^-1, respectively, in -4°C-acclimated larvae. Final specific activities of the purified enzymes were 396 and 82 units·mg protein^-1, respectively. Both enzymes were dimers with native molecular weights of 215000±18000 for glycogen phosphorylase a and 209000±15000 for glycogen phosphorylase b; the subunit molecular weight of both forms was 87000±2000. Both enzymes showed pH optima of 7.5 at 22°C and a break in the Arrhenius relationship with a two- to four-fold increase in activation energy below 10°C. Michaelis constant values for glycogen at 22°C were 0.12±0.004 mg·ml^-1 for glycogen phosphorylase a and 0.87±0.034 mg·ml^-1 for glycogen phosphorylase b; the Michaelis constant for inorganic phosphate was 6.5±0.07 mmol·l^-1 for glycogen phosphorylase a and 23.6 mmol·l^-1 for glycogen phosphorylase b. Glycogen phosphorylase b was activated by adenosine monophosphate with a K _a of 0.176±0.004 mmol·l^-1. Michaelis constant and K _a values decreased by two- to fivefold at 5°C compared with 22°C. Glycerol had a positive effect on the Michaelis constant for glycogen for glycogen phosphorylase a at intermediate concentrations (0.5 mol·l^-1) but was inhibitory to both enzyme forms at high concentrations (2 mol·l^-1). Glycerol production as a cryoprotectant in E. scudderiana larvae is facilitated by the low temperature-simulated glycogen phosphorylase b to glycogen phosphorylase a conversion and by positive effects of low temperature on the kinetic properties of glycogen phosphorylase a. Enzyme shut-down when polyol synthesis is complete appears to be aided by strong inhibitory effects of glycerol and KCl on glycogen phosphorylase b.Abbreviations E _a activation energy - GPa glycogen phosphorylase a - GPb glycogen phosphorylase b - h Hill coefficient - I ₅₀ concentration of inhibitor that reduces enzymes velocity by 50% - K _a concentration of activator that produces half-maximal activation of enzyme activity - K _m Michaelis-Menten substrate affinity constant - MW molecular weight - PEG polyethylene glycol - P_i morganic phosphate - SDS PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - V _max enzyme maximal velocity     

Regulation of the two forms of glycogen phosphorylase by cAMP and its analogs in Dictyostelium discoideum

Summary We have recently reported the existence of two forms of glycogen phosphorylase (1,4--D-glucan: orthophosphate--glucosyltransferase; EC 2.4.1.1) in Dictyostelium discoideum. During development the activity of the glycogen phosphorylase b form decreased as the activity of the a form increased. The total phosphorylase activity remained constant. The physical and kinetic properties of the Dictyostelium enzyme were similar to those of the mammalian enzyme. In mammals, cAMP regulates the conversion of the two forms by a cAMP dependent protein kinase (cAMPdPK). We report here that if cAMP is added to a single cell suspension, the Dictyostelium phosphorylase activity becomes independent of 5AMP and a 104kd peptide appears. We also show the effect of several cAMP analogs on the phosphorylase activity in these single-cell suspensions. The cAMP analogs were selected on the basis of their affinities for the membrane-bound cAMP receptor or the cytoplasmic cAMPdPK. We found that relatively low levels, 100 M, of cAMP or 2'd-cAMP added to aggregation-competent cells in shaking culture caused a loss of phosphorylase b activity and the appearance of phosphorylase a activity. The analog, 2'd-cAMP, has a high affinity for the cAMP receptor but a low affinity for the cAMPdPK. Two other analogs, Bt₂-cAMP and 8-Br-cAMP, which have low affinities for the cAMP receptor but high affinities for the cAMPdPK, required high levels (500 M) for b to a conversion. cDNAs to three cAMP-regulated genes-PL3, Dll, and D3-were used as controls in the above experiments. In order to determine if intracellular levels of cAMP were involved in the regulation of phosphorylase activity, both the phosphorylase and the PL3, D11 and D3 mRNA levels were examined in cells suspended in a glucose/albumin mixture - a medium in which adenylate cyclase is inhibited. Under these conditions, neither gene regulation nor a change in the phosphorylase b to a activity occurred in response to added extra cellular cAMP. The results suggest that an intracellular increase in cAMP is involved in the regulation of the two forms of glycogen phosphorylase in Dictyostelium.Abbreviations EGTA Ethyleneglycol-bis-(-aminoethyl ether) - N,N,N N-tetra acetic acid - SDS Sodium Dodecyl Sulfate - PAGE Polyacrylamide Gel Electrophoresis     

Characterization of a new hybrid mink-mouse clone panel: chromosomal and regional assignments of the GLO,ACY, NP,CKBB, ADH2, and ME1 loci in mink (Mustela vison)

To expand the mink map, we established a new panel consisting of 23 mink-mouse clones. On the basis of statistical criteria (Wijnen et al. 1977; Burgerhout 1978), we developed a computer program for choice of clones of the panel. Assignments of the following mink genes were achieved with the use of the hybrid panel: glyoxalase (GLO), Chromosome (Chr) 1; acetyl acylase (ACY), Chr 5; creatine phosphokinase B (CKBB), Chr 10; alcohol dehydrogenase-2 (subunit B) (ADH2), Chr 8. Using a series of clones carrying rearrangements involving mink Chr 1 and 8, we assigned the gene for ME1 to the short arm of Chr 1 and that for ADH2 to Chr 8, in the region 8p12-p24. Mapping results confirm the ones we previously obtained with a mink-Chinese hamster panel. However, by means of an improved electrophoretic technique, we revised the localization of the gene for purine nucleoside phosphorylase (NP), which has been thought to be on mink Chr 2. It is reassigned to mink Chr 10.     

Trehalose phosphorylase from Pichia fermentans and its role in the metabolism of trehalose

During a screening for novel microbial trehalose phosphorylase three Pichia strains were identified as producers of this particular enzyme that have not yet been described. To our knowledge, this is the first time that this enzyme activity has been shown in yeasts. Pichia fermentans formed trehalose phosphorylase when cultivated on a growth medium containing easily metabolizable sugers such as glucose. Addition of NaCl (0.4 M) to the medium increased the synthesis of the enzyme significantly. Production of trehalose phosphorylase was found to be growth-associated with a maximum of activity formed at the transition of the exponential to the stationary phase of growth. Trehalose phosphorylase catalyzes the phosphorolytic cleavage of trehalose, yielding glucose 1-phosphate (glucose-1-P) and glucose as products. In vitro the enzyme readily catalyzes the reverse reaction, the synthesis of trehalose from glucose and glucose-1-P. For this reaction, the enzyme of P. fermentans was found to utilize -glucose-1-P preferentially. A partially purified enzyme preparation showed a pH optimum of 6.3 for the synthesis of trehalose. The enzyme was found to be rather unstable; it was easily inactivated by dilution unless Ca²⁺ or Mn²⁺ were added. This instability is presumably caused by dissociation of the enzyme. In contrast to other yeasts, P. fermentans rapidly degraded intracellularly accumulated trehalose when the carbon source in the medium was depleted. Trehalose phosphorylase seems to be a key enzyme in the degradative pathway of trehalose in P. fermentans. Additional enzymes in this catabolic pathway of trehalose include phosphoglucomutase, glucose-6-phosphate dehydrogenase, and gluconolactonase.This contribution is part of the Ph.D. thesis of Ingrid Schick