METABOLISM OF UNIFORMLY LABELLED MALTOSE-C14 INTRODUCED INTO DETACHED WHEAT AND TOBACCO LEAVES

1956 ◽  
Vol 34 (4) ◽  
pp. 569-576 ◽  
Author(s):  
G. Krotkov ◽  
S. Rizvi
Keyword(s):  
Paper Chromatography ◽  
Specific Activity ◽  
Starch Hydrolysis ◽  
Tobacco Leaves ◽  
Metabolic Pool ◽  
Wheat Leaves ◽  
Hydrolysis Of

Uniformly labelled C14-starch was incubated in the presence of saliva yielding radioactive maltose, which was isolated by paper chromatography. When this maltose was introduced into detached tobacco and wheat leaves, it was found to be respired and transformed into other carbohydrates. There was no evidence to indicate that maltose is either a better starch former than glucose or that it acts as a specific glucose donor to fructose in the synthesis of sucrose. No dilution in the specific activity of maltose was observed even under the conditions of starch hydrolysis. This indicates that hydrolysis of starch in vivo does not result in the addition of maltose to the metabolic pool of cells.

scholarly journals A Monoacylglycerol Lipase from Mycobacterium smegmatis Involved in Bacterial Cell Interaction

2010 ◽  
Vol 192 (18) ◽  
pp. 4776-4785 ◽  
Author(s):  
Rabeb Dhouib ◽  
Françoise Laval ◽  
Frédéric Carrière ◽  
Mamadou Daffé ◽  
Stéphane Canaan
Keyword(s):  
Mycobacterium Smegmatis ◽  
Wild Type Strain ◽  
Specific Activity ◽  
Cell Interaction ◽  
Monoacylglycerol Lipase ◽  
Homologous Proteins ◽  
Dependent Activity ◽  
Hydrolysis Of

ABSTRACT MSMEG_0220 from Mycobacterium smegmatis, the ortholog of the Rv0183 gene from M. tuberculosis, recently identified and characterized as encoding a monoacylglycerol lipase, was cloned and expressed in Escherichia coli. The recombinant protein (rMSMEG_0220), which exhibits 68% amino acid sequence identity with Rv0183, showed the same substrate specificity and similar patterns of pH-dependent activity and stability as the M. tuberculosis enzyme. rMSMEG_0220 was found to hydrolyze long-chain monoacylglycerol with a specific activity of 143 ± 6 U mg−1. Like Rv0183 in M. tuberculosis, MSMEG_0220 was found to be located in the cell wall. To assess the in vivo role of the homologous proteins, an MSMEG_0220 disrupted mutant of M. smegmatis (MsΔ0220) was produced. An intriguing change in the colony morphology and in the cell interaction, which were partly restored in the complemented mutant containing either an active (ComMsΔ0220) or an inactive (ComMsΔ0220S111A) enzyme, was observed. Growth studies performed in media supplemented with monoolein showed that the ability of both MsΔ0220 and ComMsΔ0220S111A to grow in the presence of this lipid was impaired. Moreover, studies of the antimicrobial susceptibility of the MsΔ0220 strain showed that this mutant is more sensitive to rifampin and more resistant to isoniazid than the wild-type strain, pointing to a critical structural role of this enzyme in mycobacterial physiology, in addition to its function in the hydrolysis of exogenous lipids.

Depletion of Dense Granule Nucleotides during Storage of Human Platelets

1990 ◽  
Vol 63 (02) ◽  
pp. 275-278 ◽  
Author(s):  
D de Korte ◽  
C W N Gouwerok ◽  
R Fijnheer ◽  
R N I Pietersz ◽  
D Roos
Keyword(s):  
Specific Activity ◽  
Adenine Nucleotide ◽  
Human Platelets ◽  
Dense Granule ◽  
Serotonin Content ◽  
Storage Pool ◽  
Diadenosine Triphosphate ◽  
Metabolic Pool ◽  
And Storage ◽  
Hydrolysis Of

SummaryThe energy metabolism of human platelets was studied during storage of platelet concentrates. The platelets were prepared from buffy coats in PVC/DEHP bags and stored for 7 days at room temperature at a concentration of 1.0 × 109/ml with horizontal agitation. The total amount of ATP and ADP decreased with 40% during this storage. This decrease correlated with the disc-tosphere transformation associated with the loss of platelet viability. During storage, the ability to incorporate 3H-adenosine into metabolic ATP and ADP (45 min at 37° C) decreased with 50%. Via measurement of the specific activity of actin-bound ADP and the amount of incorporated radioactivity into total ATP and ADP, we calculated the content of the metabolic and storage pools of ATP and ADP. The results indicate that the decrease in adenine nucleotide levels during storage was mainly caused by a depletion of ATP and ADP from the storage pool, whereas the metabolic pool remained nearly intact. After 7 days, the ATP : ADP ratio of the storage pool had decreased from 1.0 to 0.3, indicating hydrolysis of ATP.Diadenosine-triphosphate and diadenosine-tetraphosphate (present in the storage pool) decreased with only 30%, and the serotonin content remained nearly constant. Therefore, it is unlikely that the storage pool was completely secreted. Probably, the storage pool of nucleotides serves as an internal supply for maintaining the contents of the metabolic pool of ATP and ADP during storage of platelets.

scholarly journals Inhibitory effects of histidine and their reversal. The roles of pyruvate carboxylase and N10-formyltetrahydrofolate dehydrogenase

1979 ◽  
Vol 177 (3) ◽  
pp. 833-846 ◽  
Author(s):  
M C Scrutton ◽  
I Beis
Keyword(s):  
Rat Liver ◽  
Pyruvate Carboxylase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Specific Activity ◽  
Competitive Inhibitor ◽  
Product Inhibition ◽  
Detectable Effect ◽  
Formyltetrahydrofolate Dehydrogenase ◽  
Hydrolysis Of

1. N10-Formyltetrahydrofolate dehydrogenase was purified to homogeneity from rat liver with a specific activity of 0.7–0.8 unit/mg at 25 degrees C. The enzyme is a tetramer (Mw = 413,000) composed of four similar, if not identical, substrate addition and give the Km values as 4.5 micron [(-)-N10-formyltetrahydrofolate] and 0.92 micron (NADP+) at pH 7.0. Tetrahydrofolate acts as a potent product inhibitor [Ki = 7 micron for the (-)-isomer] which is competitive with respect to N10-formyltetrahydrofolate and non-competitive with respect to NADP+. 3. Product inhibition by NADPH could not be demonstrated. This coenzyme activates N10-formyltetrahydrofolate dehydrogenase when added at concentrations, and in a ratio with NADP+, consistent with those present in rat liver in vivo. No effect of methionine, ethionine or their S-adenosyl derivatives could be demonstrated on the activity of the enzyme. 4. Hydrolysis of N10-formyltetrahydrofolate is catalysed by rat liver N10-formyltetrahydrofolate dehydrogenase at 21% of the rate of CO2 formation based on comparison of apparent Vmax. values. The Km for (-)-N10-folate is a non-competitive inhibitor of this reaction with respect to N10-formyltetrahydrofolate, with a mean Ki of 21.5 micron for the (-)-isomer. NAD+ increases the maximal rate of N10-formyltetrahydrofolate hydrolysis without affecting the Km for this substrate and decreases inhibition by tetrahydrofolate. The activator constant for NAD+ is obtained as 0.35 mM. 5. Formiminoglutamate, a product of liver histidine metabolism which accumulates in conditions of excess histidine load, is a potent inhibitor of rat liver pyruvate carboxylase, with 50% inhibition being observed at a concentration of 2.8 mM, but has no detectable effect on the activity of rat liver cytosol phosphoenolpyruvate carboxykinase measured in the direction of oxaloacetate synthesis. We propose that the observed inhibition of pyruvate carboxylase by formiminoglutamate may account in part for the toxic effect of excess histidine.

BIOSYNTHESIS OF THE COUMARINS: III. THE ROLE OF GLYCOSIDES IN THE FORMATION OF COUMARIN BY HIEROCHLOE ODORATA

1962 ◽  
Vol 40 (1) ◽  
pp. 607-618 ◽  
Author(s):  
Stewart A. Brown
Keyword(s):  
Specific Activity ◽  
Indirect Evidence ◽  
Coumaric Acid ◽  
Major Pathway ◽  
Metabolic Intermediates ◽  
Simple Extraction ◽  
Hierochloe Odorata ◽  
Hydrolysis Of

Coumarinic acid β-glucoside has been identified in Hierochloe odorata. Carbon dioxide-C14was administered to this species, and the plants were allowed to metabolize for periods up to 31 days. "Free" coumarin was recovered by simple extraction, and additional coumarin and o-coumaric acid were recovered after hydrolysis of the corresponding glucosides with emulsin. The total C14in o-coumaric acid reached a maximum in about 4 days and then declined, but the peak in the total C14of coumarin was not reached until 8 to 17 days after activation. o-Coumaryl glucoside and coumarin are thus both metabolic intermediates rather than end products, and the glucoside is formed first. This supports the orthohydroxylation theory of coumarin biosynthesis. The specific activity of coumarin liberated by emulsin hydrolysis from the glucoside was consistently lower than that of "free" coumarin. This is irreconcilable with the proposed formation of free coumarin from coumarinic acid glucoside as a major pathway in vivo unless separate pools of the glucoside exist in the plant. Indirect evidence is presented that free coumarin can exist in Hierochloe.

BIOSYNTHESIS OF THE COUMARINS: III. THE ROLE OF GLYCOSIDES IN THE FORMATION OF COUMARIN BY HIEROCHLOE ODORATA

1962 ◽  
Vol 40 (5) ◽  
pp. 607-618 ◽  
Author(s):  
Stewart A. Brown
Keyword(s):  
Specific Activity ◽  
Indirect Evidence ◽  
Coumaric Acid ◽  
Major Pathway ◽  
Metabolic Intermediates ◽  
Simple Extraction ◽  
Hierochloe Odorata ◽  
Hydrolysis Of

Coumarinic acid β-glucoside has been identified in Hierochloe odorata. Carbon dioxide-C14was administered to this species, and the plants were allowed to metabolize for periods up to 31 days. "Free" coumarin was recovered by simple extraction, and additional coumarin and o-coumaric acid were recovered after hydrolysis of the corresponding glucosides with emulsin. The total C14in o-coumaric acid reached a maximum in about 4 days and then declined, but the peak in the total C14of coumarin was not reached until 8 to 17 days after activation. o-Coumaryl glucoside and coumarin are thus both metabolic intermediates rather than end products, and the glucoside is formed first. This supports the orthohydroxylation theory of coumarin biosynthesis. The specific activity of coumarin liberated by emulsin hydrolysis from the glucoside was consistently lower than that of "free" coumarin. This is irreconcilable with the proposed formation of free coumarin from coumarinic acid glucoside as a major pathway in vivo unless separate pools of the glucoside exist in the plant. Indirect evidence is presented that free coumarin can exist in Hierochloe.

Ontogeny of gastric H(+)-K(+)-ATPase in suckling rabbits

1990 ◽  
Vol 259 (6) ◽  
pp. G913-G921 ◽  
Author(s):  
J. M. Crothers ◽  
W. W. Reenstra ◽  
J. G. Forte
Keyword(s):  
Microsomal Fraction ◽  
Apical Membrane ◽  
Specific Activity ◽  
Oxyntic Cell ◽  
Age Related ◽  
Nitrophenyl Phosphate ◽  
Weaning Age ◽  
Age Range ◽  
Hydrolysis Of

Gastric mucosal homogenates were prepared from resting and stimulated stomachs of rabbits, age 3-57 days postnatal, and fractionated by differential centrifugation. Total H(+)-K(+)-adenosinetriphosphatase (ATPase) (assayed as K(+)-dependent ouabain-insensitive hydrolysis of p-nitrophenyl phosphate) was low in the first 3 wk but rapidly accumulated between days 20 and 43. Specific activity rose eightfold from day 3 to a typically adult level of 2 mumol.mg-1.h-1 by day 43. The microsomal fraction (P3) was subfractionated on sucrose gradients (20, 27, and 33% steps or 10-40% continuous gradient). H(+)-K(+)-ATPase from P3 of resting stomachs was distributed bimodally on the continuous gradients, with activity mainly in the denser peak (or on the 33% sucrose step) before day 20, but accumulating mainly in the lighter peak (or in the lighter step-gradient fractions) after day 20. Throughout the age range tested, in vivo stimulation with histamine just before removal of the stomach caused a loss of most H(+)-K(+)-ATPase from P3 and an increase in H(+)-K(+)-ATPase in a lower-speed fraction P1. Thus, even in the 1st postnatal wk, when H(+)-K(+)-ATPase is low, most of the enzyme occurs in cells with histamine H2 receptors and all the intracellular mechanisms for fusion of oxyntic cell tubulovesicles (enriched in P3) with the apical membrane (enriched in P1). These studies delineate a 3-wk period of sharply accelerated synthesis of H(+)-K(+)-ATPase before weaning. Age-related changes in distribution of H(+)-K(+)-ATPase among microsomal density subfractions suggest maturational changes either in the intracellular partitioning of the enzyme or in properties of the membranes containing the enzyme.

PROTEIN TURNOVER IN ATTACHED WHEAT AND TOBACCO LEAVES

1964 ◽  
Vol 42 (1) ◽  
pp. 1-12 ◽  
Author(s):  
J. A. Hellebust ◽  
R. G. S. Bidwell
Keyword(s):  
Amino Acids ◽  
Protein Turnover ◽  
Specific Activity ◽  
Soluble Sugars ◽  
Turnover Rates ◽  
Protein Nitrogen ◽  
Tobacco Leaves ◽  
Long Time ◽  
Protein Amino Acids ◽  
Wheat Leaves

Attached primary and secondary wheat leaves were supplied continuously with C14O2 during daily periods of photosynthesis for 3 days. Samples were analyzed for amounts and total activities of respired carbon, soluble sugars and amino acids, protein amino acids, and protein nitrogen. By labelling all possible protein precursors to the same extent it was possible to eliminate doubts about the specific activity of carbon entering protein. Hence turnover rates could be accurately established. Because tobacco leaves last for a long time, it was possible to label their proteins, wait until soluble compounds were at a low specific activity, and then measure turnover of proteins as radioactivity in them decreased.Protein amino acid turnover rates of 0.4–0.5% per hour were obtained in rapidly growing secondary wheat leaves and 0.2–0.3% per hour in non-growing primary wheat leaves. Turnover rates of 0.15–0.2% per hour were found in expanding tobacco leaves, but little or no turnover was found in fully expanded tobacco leaves.It is suggested that protein turnover is a facet of the biochemical differentiation that accompanies development, enlargement, or change in function of an organ without concomitant net protein synthesis.

On the Preparation of Bovine α-Thrombin

1978 ◽  
Vol 39 (01) ◽  
pp. 193-200 ◽  
Author(s):  
Erwin F Workman ◽  
Roger L Lundblad
Keyword(s):  
Immobilized Enzyme ◽  
Catalytic Properties ◽  
Specific Activity ◽  
Guanidine Hydrochloride ◽  
Low Molecular Weight ◽  
Reversible Process ◽  
Gel Beads ◽  
Tissue Thromboplastin ◽  
C 50 ◽  
Hydrolysis Of

SummaryAn improved method for the preparation of bovine α-thrombin is described. The procedure involves the activation of partially purified prothrombin with tissue thromboplastin followed by chromatography on Sulfopropyl-Sephadex C-50. The purified enzyme is homogeneous on polyacrylamide discontinuous gel electrophoresis and has a specific activity toward fibrinogen of 2,200–2,700 N.I.H. U/mg. Its stability on storage in liquid media is dependent on both ionic strenght and temperature. Increasing ionic strength and decreasing temperature result in optimal stability. The denaturation of α-thrombin by guanidine hydrochloride was found to be a partially reversible process with the renatured species possessing properties similar to “aged” thrombin. In addition, the catalytic properties of a-thrombin covalently attached to agarose gel beads were also examined. The activity of the immobilized enzyme toward fibrinogen was affected to a much greater extent than was the hydrolysis of low molecular weight, synthetic substrates.

Soluble Thrombomodulin Purified from Human Urine Exhibits a Potent Anticoagulant Effect In Vitro and In Vivo

1995 ◽  
Vol 73 (05) ◽  
pp. 805-811 ◽  
Author(s):  
Yasuo Takahashi ◽  
Yoshitaka Hosaka ◽  
Hiromi Niina ◽  
Katsuaki Nagasawa ◽  
Masaaki Naotsuka ◽  
...  
Keyword(s):  
Human Urine ◽  
Specific Activity ◽  
Anticoagulant Activity ◽  
Rabbit Lung ◽  
Soluble Thrombomodulin ◽  
Molecular Weights ◽  
Dose Dependent Fashion ◽  
Dose Dependent

SummaryWe examined the anticoagulant activity of two major molecules of soluble thrombomodulin purified from human urine. The apparent molecular weights of these urinary thrombomodulins (UTMs) were 72,000 and 79,000, respectively. Both UTMs showed more potent cofactor activity for protein C activation [specific activity >5,000 thrombomodulin units (TMU)/mg] than human placental thrombomodulin (2,180 TMU/mg) and rabbit lung thrombomodulin (1,980 TMU/mg). The UTMs prolonged thrombin-induced fibrinogen clotting time (>1 TMU/ml), APTT (>5 TMU/ml), TT (>5 TMU/ml) and PT (>40 TMU/ml) in a dose-dependent fashion. These effects appeared in the concentration range of soluble thrombomodulins present in human plasma and urine. In the rat DIC model induced by thromboplastin, administration of UTMs by infusion (300-3,000 TMU/kg) restored the hematological abnormalities derived from DIC in a dose-dependent fashion. These results demonstrate that UTMs exhibit potent anticoagulant and antithrombotic activities, and could play a physiologically important role in microcirculation.

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