Phosphorylation of Ca-ATPase of Sarcoplasmic Reticulum with Different Substrates

1983 ◽  
Vol 38 (9-10) ◽  
pp. 845-847 ◽  
Author(s):  
Hector Barrabin ◽  
Leopoldo de Meis
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Low Temperatures ◽  
Hplc Analysis ◽  
Calcium Concentration ◽  
Specific Activity ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
High Calcium ◽  
High Calcium Concentration ◽  
Rate Limiting

ATP and GTP as substrate for phosphorylation of sarcoplasmic reticulum ATPase are compared. Maximal levels of phosphoenzyme are between 4.5 and 4.8 nmol per mg of protein when either substrate is used provided that phosphoenzyme hydrolysis are strongly inhibited by high calcium concentration (20 mм) and low temperatures ( 0 ° C ) . The maximal values obtained with GTP are lower than those previously reported. It is shown that this difference is due to underestimation of the specific activity of labeled nucleotides used in previous studies, as revealed by UV absorption and HPLC analysis. The dependence of the phosphoenzyme levels on calcium concentration, pH and temperature confirm previous findings indicating that ATP, but no GTP, accelerates the rate limiting step of the catalytic cycle.

scholarly journals A multi-enzyme cascade for efficient production of d-p-hydroxyphenylglycine from l-tyrosine

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Xu Tan ◽  
Sheng Zhang ◽  
Wei Song ◽  
Jia Liu ◽  
Cong Gao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Enantiomeric Excess ◽  
Efficient Production ◽  
Enzyme Cascade ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rotation Strategy ◽  
Rate Limiting

AbstractIn this study, a four-enzyme cascade pathway was developed and reconstructed in vivo for the production of d-p-hydroxyphenylglycine (D-HPG), a valuable intermediate used to produce β-lactam antibiotics and in fine-chemical synthesis, from l-tyrosine. In this pathway, catalytic conversion of the intermediate 4-hydroxyphenylglyoxalate by meso-diaminopimelate dehydrogenase from Corynebacterium glutamicum (CgDAPDH) was identified as the rate-limiting step, followed by application of a mechanism-guided “conformation rotation” strategy to decrease the hydride-transfer distance d(C6HDAP−C4NNADP) and increase CgDAPDH activity. Introduction of the best variant generated by protein engineering (CgDAPDHBC621/D120S/W144S/I169P with 5.32 ± 0.85 U·mg−1 specific activity) into the designed pathway resulted in a D-HPG titer of 42.69 g/L from 50-g/L l-tyrosine in 24 h, with 92.5% conversion, 71.5% isolated yield, and > 99% enantiomeric excess in a 3-L fermenter. This four-enzyme cascade provides an efficient enzymatic approach for the industrial production of D-HPG from cheap amino acids.

scholarly journals Mutation of Aspartate 238 in FAD Synthase Isoform 6 Increases the Specific Activity by Weakening the FAD Binding

2019 ◽  
Vol 20 (24) ◽  
pp. 6203 ◽  
Author(s):  
Piero Leone ◽  
Michele Galluccio ◽  
Stefano Quarta ◽  
Ernesto Anoz-Carbonell ◽  
Milagros Medina ◽  
...  
Keyword(s):  
Binding Sites ◽  
Specific Activity ◽  
Synthesis Rate ◽  
General Increase ◽  
Respiratory Chain Deficiency ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Acyl Coa Dehydrogenases ◽  
Rate Limiting ◽  
Fad Binding

FAD synthase (FADS, or FMN:ATP adenylyl transferase) coded by the FLAD1 gene is the last enzyme in the pathway of FAD synthesis. The mitochondrial isoform 1 and the cytosolic isoform 2 are characterized by the following two domains: the C-terminal PAPS domain (FADSy) performing FAD synthesis and pyrophosphorolysis; the N-terminal molybdopterin-binding domain (FADHy) performing a Co++/K+-dependent FAD hydrolysis. Mutations in FLAD1 gene are responsible for riboflavin responsive and non-responsive multiple acyl-CoA dehydrogenases and combined respiratory chain deficiency. In patients harboring frameshift mutations, a shorter isoform (hFADS6) containing the sole FADSy domain is produced representing an emergency protein. With the aim to ameliorate its function we planned to obtain an engineered more efficient hFADS6. Thus, the D238A mutant, resembling the D181A FMNAT “supermutant” of C. glabrata, was overproduced and purified. Kinetic analysis of this enzyme highlighted a general increase of Km, while the kcat was two-fold higher than that of WT. The data suggest that the FAD synthesis rate can be increased. Additional modifications could be performed to further improve the synthesis of FAD. These results correlate with previous data produced in our laboratory, and point towards the following proposals (i) FAD release is the rate limiting step of the catalytic cycle and (ii) ATP and FMN binding sites are synergistically connected.

Metabolic effects of ouabain in the perfused rat heart

1964 ◽  
Vol 207 (2) ◽  
pp. 347-351 ◽  
Author(s):  
Robert A. Kreisberg ◽  
John R. Williamson
Keyword(s):  
Calcium Concentration ◽  
Specific Activity ◽  
Calcium Content ◽  
Metabolic Effects ◽  
Rat Hearts ◽  
High Calcium ◽  
High Calcium Concentration ◽  
Free Glucose ◽  
Steady State Levels ◽  
Perfusion Period

Ouabain increased the uptake and oxidation of glucose-U-C14 by perfused guinea pig and rat hearts by 50%. Cardiac glycogen content remained constant and no change in glycogen specific activity was observed. The metabolic changes produced by ouabain were dependent on the calcium concentration of the perfusate. Reduction of the calcium content of the perfusate by 50% abolished the increase in C14O2 production by ouabain. The C14O2 production of hearts perfused with buffer containing twice the physiological calcium concentration was similar to that of hearts perfused with buffer containing the normal calcium concentration and ouabain. The effect of the high calcium concentration combined with ouabain was not different from that produced by either one alone. Steady-state levels of glycolytic intermediates after a 30-min perfusion period were elevated in the absence of glycogenolysis and free glucose did not accumulate intracellularly. In so far as the changes in glucose metabolism are concerned the findings are consistent with the concept that the effects of glycosides on cardiac muscle are dependent on changes in calcium transport or concentration at the site of the contractile elements.

scholarly journals Exploiting two-dimensional morphology of molybdenum oxycarbide to enable efficient catalytic dry reforming of methane

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexey Kurlov ◽  
Evgeniya B. Deeva ◽  
Paula M. Abdala ◽  
Dmitry Lebedev ◽  
Athanasia Tsoukalou ◽  
...  
Keyword(s):  
Contact Time ◽  
Specific Activity ◽  
Catalytic Performance ◽  
Dry Reforming ◽  
Dry Reforming Of Methane ◽  
Two Dimensional ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Oxygen Coverage ◽  
Rate Limiting

Abstract The two-dimensional morphology of molybdenum oxycarbide (2D-Mo2COx) nanosheets dispersed on silica is found vital for imparting high stability and catalytic activity in the dry reforming of methane. Here we report that owing to the maximized metal utilization, the specific activity of 2D-Mo2COx/SiO2 exceeds that of other Mo2C catalysts by ca. 3 orders of magnitude. 2D-Mo2COx is activated by CO2, yielding a surface oxygen coverage that is optimal for its catalytic performance and a Mo oxidation state of ca. +4. According to ab initio calculations, the DRM proceeds on Mo sites of the oxycarbide nanosheet with an oxygen coverage of 0.67 monolayer. Methane activation is the rate-limiting step, while the activation of CO2 and the C–O coupling to form CO are low energy steps. The deactivation of 2D-Mo2COx/SiO2 under DRM conditions can be avoided by tuning the contact time, thereby preventing unfavourable oxygen surface coverages.

scholarly journals A multi-enzyme cascade for efficient production of D-p-hydroxyphenylglycine from L-tyrosine

2021 ◽  
Author(s):  
Xu Tan ◽  
Sheng Zhang ◽  
Wei Song ◽  
Jia Liu ◽  
Cong Gao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Chemical Synthesis ◽  
Specific Activity ◽  
Efficient Production ◽  
Enzyme Cascade ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rotation Strategy ◽  
Rate Limiting

Abstract In this study, we designed and in vivo reconstructed a novel four-enzyme cascade pathway for the production of D-HPG, a valuable intermediate used to produce β-lactam antibiotics and for fine-chemical synthesis, from L-tyrosine. In this pathway, we identified catalytic conversion of the substrate 4-hydroxyphenylglyoxylic acid by meso-diaminopimelate dehydrogenase from Corynebacterium glutamicum (CgDAPDH) as the rate-limiting step, followed by application of a mechanism-guided “conformation rotation” strategy to decrease the hydride-transfer distance d(C6HDAP−C4NNADP) and increase CgDAPDH activity. Introduction of the best variant generated by protein engineering (CgDAPDHBC621/D120S/W144S/I169P with 5.32 ± 0.85 U·mg− 1 specific activity) into the designed pathway resulted in a D-HPG titer of 42.69 g/L from 50 g/L L-tyrosine in 24 h with 92.5% conversion and > 99% ee in a 3-L fermenter, representing the highest reported D-HPG titer to date. This four-enzyme cascade provides a novel and effective enzymatic approach to industrial production of D-HPG from cheap amino acids.

Ornithine Decarboxylase Activity in Cultured Endothelial Cells Stimulated by Serum, Thrombin and Serotonin

1978 ◽  
Vol 39 (02) ◽  
pp. 496-503 ◽  
Author(s):  
P A D’Amore ◽  
H B Hechtman ◽  
D Shepro
Keyword(s):  
Endothelial Cells ◽  
Ornithine Decarboxylase ◽  
Decarboxylase Activity ◽  
Aortic Endothelial Cells ◽  
Ornithine Decarboxylase Activity ◽  
Rate Limiting Step ◽  
Bovine Aortic Endothelial Cells ◽  
Limiting Step ◽  
Rate Limiting ◽  
Serum Serotonin

SummaryOrnithine decarboxylase (ODC) activity, the rate-limiting step in the synthesis of polyamines, can be demonstrated in cultured, bovine, aortic endothelial cells (EC). Serum, serotonin and thrombin produce a rise in ODC activity. The serotonin-induced ODC activity is significantly blocked by imipramine (10-5 M) or Lilly 11 0140 (10-6M). Preincubation of EC with these blockers together almost completely depresses the 5-HT-stimulated ODC activity. These observations suggest a manner by which platelets may maintain EC structural and metabolic soundness.

Dynamics of hepatic and peripheral insulin effects suggest common rate-limiting step in vivo

Diabetes ◽  
1993 ◽  
Vol 42 (2) ◽  
pp. 296-306 ◽  
Author(s):  
D. C. Bradley ◽  
R. A. Poulin ◽  
R. N. Bergman
Keyword(s):  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting
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