scholarly journals Studies on ligand binding to bovine liver uridine diphosphate glucose pyrophosphorylase

1976 ◽  
Vol 159 (1) ◽  
pp. 65-70 ◽  
Author(s):  
R A J Stevens ◽  
C F Phelps
Keyword(s):  
Ligand Binding ◽  
Metal Ion ◽  
Bovine Liver ◽  
Uridine Diphosphate ◽  
Uridine Diphosphate Glucose ◽  
Substrate Analogue ◽  
Diphosphate Glucose ◽  
Enzyme Intermediate

A procedure for the preparation of crystalline UDP-glucose pyrophosphorylase is described. K(s) values for UDP-glucose and UTP were determined as 7 and 20 muM respectively, the latter being confirmed by three methods. By assuming an octameric structure, 1 mol of enzyme subunit bound 1 mol of substrate. The metal-ion activator, Mg2+, did not affect the equilibrium between nucleotide and enzyme. A substrate analogue, alphabeta-methylene-UTP, was synthesized and had the same K(s) value as UTP. In its presence, the K(s) for glucose 1-phosphate decreased by two orders of magnitude, thus confirming a compulsory binding order and excluding an uridylated enzyme intermediate. The results are discussed with respect to their implications in vivo.

HISTOCHEMICAL INVESTIGATION OF THE EFFECTS OF OESTROGEN, PROGESTERONE AND RELAXIN ON GLYCOGEN, AMYLOPHOSPHORYLASE, TRANSGLYCOSYLASE AND URIDINE DIPHOSPHATE GLUCOSE-GLYCOGEN TRANSFERASE IN UTERI OF MICE

1965 ◽  
Vol 32 (2) ◽  
pp. 245-257 ◽  
Author(s):  
KATHLEEN HALL
Keyword(s):  
Enzyme Activities ◽  
Glycogen Synthesis ◽  
Glandular Epithelium ◽  
Uridine Diphosphate ◽  
Phosphorylase Activity ◽  
Uridine Diphosphate Glucose ◽  
Ovariectomized Mice ◽  
Diphosphate Glucose ◽  
Arterial Muscle

SUMMARY (1) The effects of combinations of oestrogen, progesterone and relaxin on glycogen content, and on amylophosphorylase, transglycosylase and uridine diphosphate glucose-glycogen glucosyl transferase activities in the corpus uteri of intact and ovariectomized virgin mice were investigated by histochemical techniques. (2) Glycogen and the enzyme activities were localized to myometrial and arterial muscle fibres, mobilized leucocytes when present, and luminal and glandular epithelium. Transglycosylase activity was not found in glandular epithelium and no information was obtained about UDPG-glycogen synthesis in epithelium or in leucocytes; otherwise the distribution of the three activities appeared to be similar. (3) In untreated ovariectomized mice no glycogen was detected in vivo and phosphorylase activity was low. In untreated intact mice little histochemically detectable glycogen was found in myometrial muscle at any stage of the cycle and almost no UDPG-synthesized glycogen; amylophosphorylase activity appeared to be increased during pro-oestrus and oestrus. (4) Oestrogen produced increased amounts of glycogen in vivo and stimulated phosphorylase activity in both muscle layers in intact and ovariectomized mice; UDPG-glycogen synthesis was probably also increased. (5) Relaxin had no detectable effect on myometrial glycogen or on phosphorylase activity in non-primed ovariectomized mice, but both were increased when relaxin was given to oestrogen-primed ovariectomized mice or to intact mice at pro-oestrus or oestrus. Only small increases were detected in UDPG-glycogen synthesis. (6) In both intact and ovariectomized oestrogen-primed mice progesterone had a differential action on the two layers of the myometrium: it increased both glycogenolysis and phosphorylase activity in the longitudinal fibres, but inhibited phosphorylase activity in the circular fibres without resulting in glycogen synthesis in vivo. Results on UDPG-glycogen synthesis were inconclusive. Simultaneous administration of relaxin prevented the inhibitory action of progesterone on glycogen and phosphorylase activity in the circular muscle layer and UDPG-glycogen synthesis was also high in these mice. (7) No consistent effects of the hormones were detected on glycogen or enzyme activities in arterial muscle. (8) The histochemical tests visualized total phosphorylase activity but gave no information about hormonal influence on phosphorylase a and b ratios.

scholarly journals Half-sites oxidation of bovine liver uridine diphosphate glucose dehydrogenase

1978 ◽  
Vol 173 (2) ◽  
pp. 701-704 ◽  
Author(s):  
J S Franzen ◽  
P Marchetti ◽  
R Ishman ◽  
J Ashcom
Keyword(s):  
Catalytic Activity ◽  
Glucose Dehydrogenase ◽  
Bovine Liver ◽  
Thiol Group ◽  
Catalytic Site ◽  
Uridine Diphosphate ◽  
Thiol Groups ◽  
Irreversible Denaturation ◽  
Diphosphate Glucose ◽  
Uridine Diphosphate Glucose Dehydrogenase

6,6-Dithiodinicotinate shows half-of-the-sites reactivity towards the six catalytic-site thiol groups of bovine liver UDP-glucose dehydrogenase. The reagent introduces three intrasubunit disulphide linkages between catalytic-site thiol groups and non-catalytic-site thiol groups and abrogates 60% of the catalytic activity of the hexameric enzyme; excess 2-mercaptoethanol rapidly restores full catalytic activity. These results show the half-of-the-sites behaviour of the enzyme with the reagent and the presence of a non-catalytic-site thiol group capable of forming a disulphide linkage with a catalytic-site thiol group on the same subunit without irreversible denaturation.

scholarly journals Studies on glycogen synthesis in pigeon liver homogenates. Glycogen synthesis from glucose monophosphates and uridine diphosphate glucose

1967 ◽  
Vol 105 (2) ◽  
pp. 515-519 ◽  
Author(s):  
V. N. Nigam
Keyword(s):  
Time Course ◽  
Initial Rate ◽  
Glycogen Synthesis ◽  
Uridine Diphosphate ◽  
Cytoplasmic Fraction ◽  
Uridine Diphosphate Glucose ◽  
Diphosphate Glucose ◽  
Liver Homogenates ◽  
Pigeon Liver ◽  
Glycogen Formation

Comparative time-course studies of glycogen synthesis from glucose 6-phosphate, glucose 1-phosphate and UDP-glucose show that glucose 1-phosphate forms glycogen at an initial rate faster than that obtained with glucose 6-phosphate and UDP-glucose. After 5min. the rates from glucose monophosphates are considerably slower. 2,4-Dinitrophenol decreases glycogen synthesis from both glucose monophosphates, whereas arsenate and EDTA increase glycogen synthesis from glucose 1-phosphate and inhibit the reaction from glucose 6-phosphate, galactose and galactose 1-phosphate. Mitochondria-free pigeon liver cytoplasmic fraction forms less glycogen from glucose monophosphates than does the whole homogenate. 2-Deoxyglucose 6-phosphate inhibits glycogen synthesis from glucose monophosphates. Glycogen formation from UDP-glucose is relatively unaffected by dinitrophenol, by arsenate, by EDTA, by 2-deoxyglucose 6-phosphate and by the removal of mitochondria from the whole homogenate.

scholarly journals Enzymic transfer of glucose and xylose from uridine diphosphate glucose and uridine diphosphate xylose to bilirubin by untreated and digitonin-activated preparations from rat liver

1972 ◽  
Vol 129 (3) ◽  
pp. 619-633 ◽  
Author(s):  
J. Fevery ◽  
P. Leroy ◽  
K. P. M. Heirwegh
Keyword(s):  
Enzyme Activities ◽  
Metal Ion ◽  
Glucuronic Acid ◽  
Uridine Diphosphate ◽  
No Formation ◽  
Cell Extracts ◽  
Straight Line ◽  
Standard Assay ◽  
Diphosphate Glucose ◽  
Liver Homogenates

1. Digitonin-treated and untreated homogenates, cell extracts and washed microsomal preparations from liver of Wistar R rats are capable of transferring sugar from UDP-glucose or UDP-xylose to bilirubin. No formation of bilirubin glycosides occurred with UDP-galactose or d-glucose, d-xylose or d-glucuronic acid as the sources of sugar. 2. Procedures to assay digitonin-activated and unactivated bilirubin UDP-glucosyltransferase and bilirubin UDP-xylosyltransferase were developed. 3. In digitonin-activated microsomal preparations the transferring enzymes had the following properties. Both enzyme activities were increased 2.5-fold by pretreatment with digitonin. They were optimum at pH6.6–7.2. Michaelis–Menten kinetics were followed with respect to UDP-glucose. In contrast, double-reciprocal plots of enzyme activity against the concentration of UDP-xylose showed two intersecting straight-line sections corresponding to concentration ranges where either bilirubin monoxyloside was formed (at low UDP-xylose concentrations) or where mixtures of both the mono- and di-xyloside were synthesized (at high UDP-xylose concentrations). Both enzyme activities were stimulated by Mg2+; Ca2+ was slightly less, and Mn2+ slightly more, stimulatory than Mg2+. Of the activities found in standard assay systems containing Mg2+, 58–78% (substrate UDP-glucose) and 0–38% (substrate UDP-xylose) were independent of added bivalent metal ion. Double-reciprocal plots of the Mg2+-dependent activities against the concentration of added Mg2+ were linear. 4. In comparative experiments the relative activities of liver homogenates obtained with UDP-glucuronic acid, UDP-glucose and UDP-xylose were 1:1.5:2.7 for untreated preparations and 1:0.29:0.44 after activation with digitonin. 5. Bilirubin UDP-glucuronyltransferase was protected against denaturation by human serum albumin, whereas bilirubin UDP-xylosyltransferase was not. 6. Digitonin-treated and untreated liver homogenates from Gunn rats were inactive in transferring sugar to bilirubin from UDP-glucuronic acid (in agreement with the work of others), UDP-glucose or UDP-xylose.

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