Refinement of the Kinetic Model of the 2-[14C]Deoxyglucose Method to Incorporate Effects of Intracellular Compartmentation in Brain

1989 ◽  
Vol 9 (3) ◽  
pp. 290-303 ◽  
Author(s):  
K. Schmidt ◽  
G. Lucignani ◽  
K. Mori ◽  
T. Jay ◽  
E. Palombo ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Rate Constant ◽  
Step Process ◽  
Precursor Pool ◽  
Physiological Conditions ◽  
Dg Method ◽  
Yield Values ◽  
Rate Limiting ◽  
Hydrolysis Of ◽  
Deoxyglucose Method

A translocase to transport hexose phosphate formed in the cytosol into the cisterns of the endoplasmic reticulum, where the phosphatase resides, is absent in brain (Fishman and Karnovsky, 1986). 2-Deoxyglucose-6-phosphate (DG-6-P) may therefore have limited access to glucose-6-phosphatase (G-6-Pase), and transport of the DG-6-P across the endoplasmic reticular membrane may be rate limiting to its dephosphorylation. To take this compartmentation into account, a five-rate constant (5K) model was developed to describe the kinetic behavior of 2-deoxyglucose (DG) and its phosphorylated product in brain. Loss of DG-6-P was modeled as a two-step process: (a) transfer of DG-6-P from the cytosol into the cisterns of the endoplasmic reticulum; (b) hydrolysis of DG-6-P by G-6-Pase and subsequent return of the free DG to the precursor pool. Local CMRglc (LCMRglc) was calculated in the rat on the basis of this model and compared with values calculated on the basis of the three-rate constant (3K) and the four–rate constant (4K) models of the DG method. The results show that under normal physiological conditions all three models yield values of LCMRglc that are essentially equivalent for experimental periods between 25 and 45 min. Therefore, the simplest model, the 3K model, is sufficient. For experimental periods from 60 to 120 min, the 4K and 5K models do not correct completely for loss of product, but the 5K model does yield estimates of LCMRglc that are closer to the values at 45 min than those obtained with the 3K and 4K models.

Hydrolysis of a monocyclic oxyphosphorane containing a five-membered ring

1991 ◽  
Vol 69 (12) ◽  
pp. 2075-2083 ◽  
Author(s):  
Glenn H. McGall ◽  
Robert A. McClelland
Keyword(s):  
Rate Constant ◽  
Methoxy Group ◽  
Ring Closure ◽  
Rate Limiting Step ◽  
Acid Base Equilibrium ◽  
Reaction With Water ◽  
Effective Molarity ◽  
Rate Limiting ◽  
Hydrolysis Of ◽  
Good Agreement

A kinetic study is reported for the hydrolysis of 2,2-diphenyl-2-methoxy-1,3,2-dioxaphospholane 1. This phosphorane exists in aqueous solution in a pseudo acid–base equilibrium with an observable phosphonium ion, the ring-opened (2′-hydroxyethoxy)diphenylmethoxyphosphonium ion 5. The equilibrium constant Ka ([1][H+]/[5]) is 9 × 10−9, values determined by kinetic and spectroscopic methods being in good agreement. This phosphonium ion is, however, not involved in the overall hydrolysis reaction, which proceeds via the thermodynamically less stable cyclic five-membered phosphonium ion derived by loss of the exocyclic methoxy group from the phosphorane, the 2,2-diphenyl-1,3,2-dioxaphospholan-2-ylium ion 6. This route for the overall hydrolysis is established by analysis of the products, and by the observation that the rate constant for the disappearance of 5 in acid solutions is 40 000 times greater than that for an analog that differs only in not being able to cyclize, the (2′-methoxyethoxy)diphenylmethoxyphosphonium ion 7. At all pH, the phosphorane 1 and the ring-opened phosphonium ion 5 exist in equilibrium, and the rate-limiting step in the overall hydrolysis is the cleavage of the exocyclic methoxy group to give the cyclic phosphonium ion 6, which is rapidly converted to products by reaction with water. The actual equilibration reaction involving 1 and 5 cannot be observed at any pH, even with stopped-flow spectroscopy. The non-catalyzed ring closure of the phosphonium ion 5 reforming the phosphorane 1 occurs with a rate constant of 200–500 s−1, corresponding to an effective molarity of (2–5) × 107 M for the intramolecular hydroxy group in this reaction. The rate-limiting exocyclic cleavage is assisted by H+, with a very large rate constant 2 × 109 M−1 s−1. Catalysis by general acids is also observed. The Brønsted plot has a slope α of 1.0 for the weaker acids, with a break for acids with pKa < 3. This "Eigen"-type behavior is proposed to arise from a transition state with little phosphonium ion character, in which the proton is almost completely transferred for the weaker acids. Key words: phosphorane, phosphate, phosphonium, hydrolysis.

scholarly journals Type 1 polyisoprenoid diphosphate phosphatase modulates geranylgeranyl-mediated control of HMG CoA reductase and UBIAD1

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Rania Elsabrouty ◽  
Youngah Jo ◽  
Seonghwan Hwang ◽  
Dong-Jae Jun ◽  
Russell A DeBose-Boyd
Keyword(s):  
Endoplasmic Reticulum ◽  
Vitamin K ◽  
Feedback Mechanism ◽  
Hmg Coa Reductase ◽  
Golgi Transport ◽  
Coa Reductase ◽  
Rate Limiting ◽  
Hydrolysis Of ◽  
Alcohol Derivative

UbiA prenyltransferase domain-containing protein-1 (UBIAD1) utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K2 subtype menaquinone-4. The prenyltransferase has emerged as a key regulator of sterol-accelerated, endoplasmic reticulum (ER)-associated degradation (ERAD) of HMG CoA reductase, the rate-limiting enzyme in synthesis of cholesterol and nonsterol isoprenoids including GGpp. Sterols induce binding of UBIAD1 to reductase, inhibiting its ERAD. Geranylgeraniol (GGOH), the alcohol derivative of GGpp, disrupts this binding and thereby stimulates ERAD of reductase and translocation of UBIAD1 to Golgi. We now show that overexpression of Type 1 polyisoprenoid diphosphate phosphatase (PDP1), which dephosphorylates GGpp and other isoprenyl pyrophosphates to corresponding isoprenols, abolishes protein geranylgeranylation as well as GGOH-induced ERAD of reductase and Golgi transport of UBIAD1. Conversely, these reactions are enhanced in the absence of PDP1. Our findings indicate PDP1-mediated hydrolysis of GGpp significantly contributes to a feedback mechanism that maintains optimal intracellular levels of the nonsterol isoprenoid.

Product Release is Not the Rate-Limiting Step during Cytochalasin B-Induced ATPase Activity of Monomeric Actin

1991 ◽  
Vol 46 (1-2) ◽  
pp. 139-144 ◽  
Author(s):  
Peter Dancker ◽  
Lore Hess ◽  
Karl Ritter
Keyword(s):  
Steady State ◽  
Atpase Activity ◽  
Rate Constant ◽  
Cytochalasin B ◽  
Single Turnover ◽  
Rate Limiting Step ◽  
Hydrolysis Products ◽  
Limiting Step ◽  
Rate Limiting ◽  
Hydrolysis Of

Abstract Under conditions where cytochalasin B induces ATPase activity of monomeric actin (0.3 mᴍ MgCl2, 1 mᴍ EGTA , 30 (μᴍ cytochalasin B, 1 mᴍ ATP) the rate constant of the ex­change of actin-bound ε-ATP for free ATP is about 4 -6 times faster than steady state ATPase activity. When a stoichiometric ATP -actin complex is extracted with PCA (single turnover ex­periment) the apparent rate constant of Pi generation is not faster than steady state ATPase activity. -The experiments suggest that the hydrolysis of actin-bound ATP and not the subse­quent release of hydrolysis products is rate-limiting during cytochalasin-induced ATPase activi­ty of actin.

scholarly journals Kinetics and Mechanistic Study of Hydrolysis of Adenosine Monophosphate Disodium Salt (AMPNa2) in Acidic and Alkaline Media

2019 ◽  
Vol 17 (1) ◽  
pp. 544-556
Author(s):  
Yoke-Leng Sim ◽  
Beljit Kaur
Keyword(s):  
Rate Constant ◽  
Adenosine Monophosphate ◽  
Disodium Salt ◽  
Second Order ◽  
Information Storage ◽  
Alkaline Media ◽  
Basic Medium ◽  
Mechanistic Study ◽  
Phosphate Ester ◽  
Hydrolysis Of

AbstractPhosphate ester hydrolysis is essential in signal transduction, energy storage and production, information storage and DNA repair. In this investigation, hydrolysis of adenosine monophosphate disodium salt (AMPNa2) was carried out in acidic, neutral and alkaline conditions of pH ranging between 0.30-12.71 at 60°C. The reaction was monitored spectrophotometrically. The rate ranged between (1.20 ± 0.10) × 10-7 s-1 to (4.44 ± 0.05) × 10-6 s-1 at [NaOH] from 0.0008 M to 1.00M recorded a second-order base-catalyzed rate constant, kOH as 4.32 × 10-6 M-1 s-1. In acidic conditions, the rate ranged between (1.32 ± 0.06) × 10-7 s-1 to (1.67 ± 0.10) × 10-6 s-1 at [HCl] from 0.01 M to 1.00 M. Second-order acid-catalyzed rate constant, kH obtained was 1.62 × 10-6 M-1 s-1. Rate of reaction for neutral region, k0 was obtained from graphical method to be 10-7 s-1. Mechanisms were proposed to involve P-O bond cleavage in basic medium while competition between P-O bond and N-glycosidic cleavage was observed in acidic medium. In conclusion, this study has provided comprehensive information on the kinetic parameters and mechanism of cleavage of AMPNa2 which mimicked natural AMP cleavage and the action of enzymes that facilitate its cleavage.

scholarly journals Maturation of the Na,K-ATPase in the Endoplasmic Reticulum in Health and Disease

2021 ◽  
Author(s):  
Vitalii Kryvenko ◽  
Olga Vagin ◽  
Laura A. Dada ◽  
Jacob I. Sznajder ◽  
István Vadász
Keyword(s):  
Endoplasmic Reticulum ◽  
Intracellular Signaling ◽  
Loss Of Function ◽  
Α Subunit ◽  
Rate Limiting Step ◽  
Atpase Subunits ◽  
A Cell ◽  
Health And Disease ◽  
And Function ◽  
Rate Limiting

Abstract The Na,K-ATPase establishes the electrochemical gradient of cells by driving an active exchange of Na+ and K+ ions while consuming ATP. The minimal functional transporter consists of a catalytic α-subunit and a β-subunit with chaperon activity. The Na,K-ATPase also functions as a cell adhesion molecule and participates in various intracellular signaling pathways. The maturation and trafficking of the Na,K-ATPase include co- and post-translational processing of the enzyme in the endoplasmic reticulum (ER) and the Golgi apparatus and subsequent delivery to the plasma membrane (PM). The ER folding of the enzyme is considered as the rate-limiting step in the membrane delivery of the protein. It has been demonstrated that only assembled Na,K-ATPase α:β-complexes may exit the organelle, whereas unassembled, misfolded or unfolded subunits are retained in the ER and are subsequently degraded. Loss of function of the Na,K-ATPase has been associated with lung, heart, kidney and neurological disorders. Recently, it has been shown that ER dysfunction, in particular, alterations in the homeostasis of the organelle, as well as impaired ER-resident chaperone activity may impede folding of Na,K-ATPase subunits, thus decreasing the abundance and function of the enzyme at the PM. Here, we summarize our current understanding on maturation and subsequent processing of the Na,K-ATPase in the ER under physiological and pathophysiological conditions. Graphic Abstract

scholarly journals Resynthesis of phosphatidylinositol in permeabilized neutrophils following phospholipase Cβ activation: transport of the intermediate, phosphatidic acid, from the plasma membrane to the endoplasmic reticulum for phosphatidylinositol resynthesis is not dependent on soluble lipid carriers or vesicular transport

1999 ◽  
Vol 341 (2) ◽  
pp. 435-444 ◽  
Author(s):  
Jacqueline WHATMORE ◽  
Claudia WIEDEMANN ◽  
Pennti SOMERHARJU ◽  
Philip SWIGART ◽  
Shamshad COCKCROFT
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Vesicular Transport ◽  
Human Neutrophils ◽  
Permeabilized Cells ◽  
Transfer Protein ◽  
Membrane Contact ◽  
Phosphatidylinositol Transfer ◽  
Pi Hydrolysis ◽  
Hydrolysis Of

Receptor-mediated phospholipase C (PLC) hydrolysis of phosphoinositides is accompanied by the resynthesis of phosphatidylinositol (PI). Hydrolysis of phosphoinositides occurs at the plasma membrane, and the resulting diacylglycerol (DG) is converted into phosphatidate (PA). Two enzymes located at the endoplasmic reticulum (ER) function sequentially to convert PA back into PI. We have established an assay whereby the resynthesis of PI could be followed in permeabilized cells. In the presence of [γ-32P]ATP, DG generated by PLC activation accumulates label when converted into PA. The 32P-labelled PA is subsequently converted into labelled PI. The formation of labelled PI reports the arrival of labelled PA from the plasma membrane to the ER. Cytosol-depleted, permeabilized human neutrophils are capable of PI resynthesis following stimulation of PLCβ (in the presence of phosphatidylinositol-transfer protein), provided that CTP and inositol are also present. We also found that wortmannin, an inhibitor of endocytosis, or cooling the cells to 15 °C did not stop PI resynthesis. We conclude that PI resynthesis is dependent neither on vesicular transport mechanisms nor on freely diffusible, soluble transport proteins. Phosphatidylcholine-derived PA generated by the ADP-ribosylation-factor-stimulated phospholipase D pathway was found to accumulate label, reflecting the rapid cycling of PA to DG, and back. This labelled PA was not converted into PI. We conclude that PA derived from the PLC pathway is selected for PI resynthesis, and its transfer to the ER could be membrane-protein-mediated at sites of close membrane contact.

scholarly journals Retrograde trafficking of Argonaute 2 acts as a rate-limiting step for de novo miRNP formation on endoplasmic reticulum–attached polysomes in mammalian cells

2020 ◽  
Vol 3 (2) ◽  
pp. e201800161 ◽  
Author(s):  
Mainak Bose ◽  
Susanta Chatterjee ◽  
Yogaditya Chakrabarty ◽  
Bahnisikha Barman ◽  
Suvendra N Bhattacharyya
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
De Novo ◽  
Mirna Biogenesis ◽  
Argonaute 2 ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Cellular Context ◽  
Subcellular Compartmentalization ◽  
Rate Limiting

microRNAs are short regulatory RNAs in metazoan cells. Regulation of miRNA activity and abundance is evident in human cells where availability of target messages can influence miRNA biogenesis by augmenting the Dicer1-dependent processing of precursors to mature microRNAs. Requirement of subcellular compartmentalization of Ago2, the key component of miRNA repression machineries, for the controlled biogenesis of miRNPs is reported here. The process predominantly happens on the polysomes attached with the endoplasmic reticulum for which the subcellular Ago2 trafficking is found to be essential. Mitochondrial tethering of endoplasmic reticulum and its interaction with endosomes controls Ago2 availability. In cells with depolarized mitochondria, miRNA biogenesis gets impaired, which results in lowering of de novo–formed mature miRNA levels and accumulation of miRNA-free Ago2 on endosomes that fails to interact with Dicer1 and to traffic back to endoplasmic reticulum for de novo miRNA loading. Thus, mitochondria by sensing the cellular context regulates Ago2 trafficking at the subcellular level, which acts as a rate-limiting step in miRNA biogenesis process in mammalian cells.

scholarly journals Dissection of the asynchronous transport of intestinal microvillar hydrolases to the cell surface.

1988 ◽  
Vol 106 (6) ◽  
pp. 1853-1861 ◽  
Author(s):  
B Stieger ◽  
K Matter ◽  
B Baur ◽  
K Bucher ◽  
M Höchli ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Intracellular Transport ◽  
Colon Adenocarcinoma ◽  
Subcellular Fractionation ◽  
Adenocarcinoma Cell Line ◽  
Dipeptidylpeptidase Iv ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Novel subcellular fractionation procedures and pulse-chase techniques were used to study the intracellular transport of the microvillar membrane hydrolases sucrase-isomaltase and dipeptidylpeptidase IV in the differentiated colon adenocarcinoma cell line Caco-2. The overall rate of transport to the cell surface was two fold faster for dipeptidylpeptidase IV than for sucrase-isomaltase, while no significant differences were observed in transport rates from the site of complex glycosylation to the brush border. The delayed arrival of sucrase-isomaltase in the compartment where complex glycosylation occurs was only in part due to exit from the endoplasmic reticulum. A major slow-down could be ascribed to maturation in and transit of this enzyme through the Golgi apparatus. These results suggest that the observed asynchronism is due to more than one rate-limiting step along the rough endoplasmic reticulum to trans-Golgi pathway.

scholarly journals Kinetics of the hydrolysis of N-benzoyl-l-serine methyl ester catalysed by bromelain and by papain. Analysis of modifier mechanisms by lattice nomography, computational methods of parameter evaluation for substrate-activated catalyses and consequences of postulated non-productive binding in bromelain- and papain-catalysed hydrolyses

1974 ◽  
Vol 141 (2) ◽  
pp. 365-381 ◽  
Author(s):  
Christopher W. Wharton ◽  
Athel Cornish-Bowden ◽  
Keith Brocklehurst ◽  
Eric M. Crook
Keyword(s):  
Methyl Ester ◽  
Dissociation Constant ◽  
Computational Methods ◽  
Rate Constant ◽  
Guanidine Hydrochloride ◽  
Ester Hydrolysis ◽  
Binary Complex ◽  
Substrate Activation ◽  
Hydrolysis Of ◽  
Ester Substrate

1. N-Benzoyl-l-serine methyl ester was synthesized and evaluated as a substrate for bromelain (EC 3.4.22.4) and for papain (EC 3.4.22.2). 2. For the bromelain-catalysed hydrolysis at pH7.0, plots of [S0]/vi (initial substrate concn./initial velocity) versus [S0] are markedly curved, concave downwards. 3. Analysis by lattice nomography of a modifier kinetic mechanism in which the modifier is substrate reveals that concave-down [S0]/vi versus [S0] plots can arise when the ratio of the rate constants that characterize the breakdown of the binary (ES) and ternary (SES) complexes is either less than or greater than 1. In the latter case, there are severe restrictions on the values that may be taken by the ratio of the dissociation constants of the productive and non-productive binary complexes. 4. Concave-down [S0]/vi versus [S0] plots cannot arise from compulsory substrate activation. 5. Computational methods, based on function minimization, for determination of the apparent parameters that characterize a non-compulsory substrate-activated catalysis are described. 6. In an attempt to interpret the catalysis by bromelain of the hydrolysis of N-benzoyl-l-serine methyl ester in terms of substrate activation, the general substrate-activation model was simplified to one in which only one binary ES complex (that which gives rise directly to products) can form. 7. In terms of this model, the bromelain-catalysed hydrolysis of N-benzoyl-l-serine methyl ester at pH7.0, I=0.1 and 25°C is characterized by Km1 (the dissociation constant of ES)=1.22±0.73mm, k (the rate constant for the breakdown of ES to E+products, P)=1.57×10-2±0.32×10-2s-1, Ka2 (the dissociation constant that characterizes the breakdown of SES to ES and S)=0.38±0.06m, and k′ (the rate constant for the breakdown of SES to E+P+S)=0.45±0.04s-1. 8. These parameters are compared with those in the literature that characterize the bromelain-catalysed hydrolysis of α-N-benzoyl-l-arginine ethyl ester and of α-N-benzoyl-l-arginine amide; Km1 and k for the serine ester hydrolysis are somewhat similar to Km and kcat. for the arginine amide hydrolysis and Kas and k′ for the serine ester hydrolysis are somewhat similar to Km and kcat. for the arginine ester hydrolysis. 9. A previous interpretation of the inter-relationships of the values of kcat. and Km for the bromelain-catalysed hydrolysis of the arginine ester and amide substrates is discussed critically and an alternative interpretation involving substantial non-productive binding of the arginine amide substrate to bromelain is suggested. 10. The parameters for the bromelain-catalysed hydrolysis of the serine ester substrate are tentatively interpreted in terms of non-productive binding in the binary complex and a decrease of this type of binding by ternary complex-formation. 11. The Michaelis parameters for the papain-catalysed hydrolysis of the serine ester substrate (Km=52±4mm, kcat.=2.80±0.1s-1 at pH7.0, I=0.1, 25.0°C) are similar to those for the papain-catalysed hydrolysis of methyl hippurate. 12. Urea and guanidine hydrochloride at concentrations of 1m have only small effects on the kinetic parameters for the hydrolysis of the serine ester substrate catalysed by bromelain and by papain.

The hydrolysis and biogas production of complex cellulosic substrates using three anaerobic biomass sources

2013 ◽  
Vol 67 (2) ◽  
pp. 293-298 ◽  
Author(s):  
C. Keating ◽  
D. Cysneiros ◽  
T. Mahony ◽  
V. O'Flaherty
Keyword(s):  
Biogas Production ◽  
Positive Role ◽  
Diverse Range ◽  
Factors Affecting ◽  
Rate Limiting Step ◽  
Solid Substrates ◽  
Different Temperatures ◽  
Anaerobic Biomass ◽  
Rate Limiting ◽  
Hydrolysis Of

In this study, the ability of various sludges to digest a diverse range of cellulose and cellulose-derived substrates was assessed at different temperatures to elucidate the factors affecting hydrolysis. For this purpose, the biogas production was monitored and the specific biogas activity (SBA) of the sludges was employed to compare the performance of three anaerobic sludges on the degradation of a variety of complex cellulose sources, across a range of temperatures. The sludge with the highest performance on complex substrates was derived from a full-scale bioreactor treating sewage at 37 °C. Hydrolysis was the rate-limiting step during the degradation of complex substrates. No activity was recorded for the synthetic cellulose compound carboxymethylcellulose (CMC) using any of the sludges tested. Increased temperature led to an increase in hydrolysis rates and thus SBA values. The non-granular nature of the mesophilic sludge played a positive role in the hydrolysis of solid substrates, while the granular sludges proved more effective on the degradation of soluble compounds.

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