A Spectrophotometric Method to Measure Enzymatic Activity in Reactions That Generate Inorganic Pyrophosphate

Upson, Rosalyn H.; Haugland, Rosaria P.; Malekzadeh, Mohammad N.; Haugland, Richard P.

doi:10.1006/abio.1996.0479

Cited by 83 publications

(67 citation statements)

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“…Furthermore, the affinity for G3P binding in the present work (K d of 0.7 μM) is 5-fold greater than that previously published (K d of 3.54 μM) (15). In the current experiments, binding measurements were performed immediately using freshly purified protein, whereas the previous measurements were performed with protein that had been stored in detergent solution for several days, which may have led to a partial loss of the integrity of the transporter.Temperature dependence of transport reaction rates has been observed before for other transporter proteins (26,29,34), although the significance in many cases was not explained in terms of the transport mechanism. Electrophysiology measurements of the voltage dependence of the L-asparagine transporter in endoderm cells from Xenopus also showed that V max increased with temperature but the K m value remained unaltered (25).…”

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confidence: 77%

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Kinetic Evidence Is Consistent with the Rocker-Switch Mechanism of Membrane Transport by GlpT

et al. 2007

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Secondary active transport of substrate across the cell membrane is crucial to many cellular and physiological processes. The crystal structure of one member of the secondary active transporter family, the sn-glycerol-3-phosphate (G3P) transporter (GlpT) of the inner membrane of Escherichia coli, suggests a mechanism for substrate translocation across the membrane that involves a rockerswitch-type movement of the protein. This rocker-switch mechanism makes two specific predictions with respect to kinetic behavior: the transport rate increases with the temperature, whereas the binding affinity of the transporter to a substrate is temperature-independent. In this work, we directly tested these two predictions by transport kinetics and substrate-binding experiments, integrating the data on this single system into a coherent set of observations. The transport kinetics of the physiologically relevant G3P-phosphate antiport reaction were characterized at different temperatures using both E. coli whole cells and GlpT reconstituted into proteoliposomes. Substrate-binding affinity of the transporter was measured using tryptophan fluorescence quenching in detergent solution. Indeed, the substrate transport velocity of GlpT increased dramatically with temperature. In contrast, neither the apparent Michaelis constant (K m ) nor the apparent substrate-binding dissociation constant (K d ) showed temperature dependence. Moreover, GlpT-catalyzed G3P translocation exhibited a completely linear Arrhenius function with an activation energy of 35.2 kJ mol −1 for the transporter reconstituted into proteoliposomes, suggesting that the substrate-loaded transporter is delicately poised between the inward-and outward-facing conformations. When these results are taken together, they are in agreement with a rocker-switch mechanism for GlpT.Secondary active membrane transporter proteins use an electrochemical gradient generated across the membrane by primary active transport as the driving force for substrate translocation (1,2). Over 100 families of secondary active membrane transporters have been identified to date, the largest of which is the major facilitator superfamily (MFS), 1 with over 5000 known members (3-5). The MFS, members of which are ubiquitous in all three kingdoms of life, includes many medically and pharmaceutically important transporters, such as the efflux pumps that confer resistance to antibiotics in bacteria and chemotherapeutic drugs in humans (6,7). † This work was supported in part by NIH Grant . The work of P.C.M. was supported by NIH Grant GM-24195. * To whom correspondence should be addressed. Telephone: (212) 263-8951. E-mail: wang@saturn.med.nyu.edu. ‡ New York University School of Medicine. § Johns Hopkins School of Medicine.1 Abbreviations: GlpT, glycerol-3-phosphate transporter; G3P, glycerol-3-phosphate; P i , inorganic phosphate; MFS, major facilitator superfamily; E a , activation energy; DDM, N-dodecyl-β-D-maltoside. Over the years, extensive efforts have been made to understand the molecular ...

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confidence: 77%

“…This inconsistency is probably due to the fact that the previous experiments used a commercially available assay kit (EnzChek, Invitrogen) that provides a spectrophotometric method for the quantitation of P i (34). It was found subsequently that, in addition to inorganic phosphate, the kit also detected organic phosphates, such as G3P.…”

Section: Discussionmentioning

confidence: 99%

Kinetic Evidence Is Consistent with the Rocker-Switch Mechanism of Membrane Transport by GlpT

et al. 2007

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“…We used ClustalX to construct multiple protein alignments (30), Psi-BLAST A C B (31) (www.ncbi.nlm.nih.gov/BLAST) to conduct long-range similarity searches, the PFAM (32) Enzyme Assays. IMPCT activity was assayed by measurement of pyrophosphate production using the EnzChek Pyrophosphate Assay Kit from Invitrogen (35). The recombinant IMPCT (typically 0.5-1.0 mg) was incubated at 80°C with I-1-P (0.2-1.5 mM) and CTP (0.2-1.2 mM) in 100 mM Tris⅐HCl (pH 8.0) with 5 mM MgCl 2 added, for 0.5, 1, and 2 min.…”

Section: Methodsmentioning

confidence: 99%

Genomic identification and in vitro reconstitution of a complete biosynthetic pathway for the osmolyte di- myo -inositol-phosphate

Rodionov

Kurnasov

Stec

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Di-myo-inositol 1,1 -phosphate (DIP) is a major osmoprotecting metabolite in a number of hyperthermophilic species of archaea and bacteria. Although the DIP biosynthesis pathway was previously proposed, genes encoding only two of the four required enzymes, inositol-1-phosphate synthase and inositol monophosphatase, were identified. In this study we used a comparative genomic analysis to predict two additional genes of this pathway (termed dipA and dipB) that remained missing. In Thermotoga maritima both candidate genes (in an originally misannotated locus TM1418) form an operon with the inositol-1-phosphate synthase encoding gene (TM1419). A predicted inositol-monophosphate cytidylyltransferase activity was directly confirmed for the purified product of T. maritima gene dipA cloned and expressed in Escherichia coli. The entire DIP pathway was reconstituted in E. coli by cloning of the TM1418 -TM1419 operon in pBAD expression vector and confirmed to function in the crude lysate. 31 P NMR and MS analysis revealed that DIP synthesis proceeds via a phosphorylated DIP intermediate, P-DIP, which is generated by the dipBencoded enzyme, now termed P-DIP synthase. This previously unknown intermediate is apparently converted to the final product, DIP, by an inositol monophosphatase-like phosphatase. These findings allowed us to revise the previously proposed DIP pathway. The genomic survey confirmed its presence in the species known to use DIP for osmoprotection. Among several newly identified species with a postulated DIP pathway, Aeropyrum pernix was directly proven to produce this osmolyte.comparative genomics ͉ di-myo-inositol-1,3-phosphate biosynthesis ͉ Thermotoga maritima T he most common mechanism of osmoadaptation in microorganisms involves the accumulation of specific organic osmolytes, so-called compatible solutes, amino acids, sugars, and polyols, that can be taken up from the environment or synthesized de novo (1-3). In thermophiles and hyperthermophiles, compatible solutes are generally different from those found in mesophiles (4), and many of them additionally contribute to thermoprotection.Di-myo-inositol 1,1Ј-phosphate (DIP) is one of the major compatible solutes in a number of thermophilic archaea of the genera Pyrococcus, Methanococcus, Thermococcus, and Archaeoglobus and bacteria belonging to the genera Aquifex, Rubrobacter, and Thermotoga, including Thermotoga maritima and Thermotoga neapolitana (4-7). DIP levels are highly increased at supraoptimal growth temperatures in both Methanococcus igneus and Thermotogales, suggesting that this solute also plays a thermoprotective role (6,8).Based on the study in M. igneus (9) and the reported stereochemistry of DIP (10), the pathway of DIP biosynthesis was originally proposed to occur in four steps: (i) synthesis of L-myoinositol-1-phosphate (L-I-1-P) from glucose-6-phosphate by NAD ϩ -dependent L-I-1-P synthase [inositol-1-phosphate synthase (IPS)]; (ii) hydrolysis of some of the L-I-1-P by inositol monophosphatase (IMP); (iii) coupling of the L-I-1-P with ...

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“…Coupled Spectrophotometric Assay for Acyl-CoA Synthetase Activity-Acyl-CoA synthetase activity was assayed by monitoring the inorganic pyrophosphate (PP i ) produced in the first half-reaction (28). Pyrophosphate production was assayed as phosphate using a commercial spectrophotometric assay (EnzChek® Pyrophosphate Assay Kit, Molecular Probes) in the presence of pyrophosphatase.…”

Section: Methodsmentioning

confidence: 99%

The Escherichia coli fadK (ydiD) Gene Encodes an Anerobically Regulated Short Chain Acyl-CoA Synthetase

Morgan‐Kiss¹,

Cronan

2004

Journal of Biological Chemistry

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A Spectrophotometric Method to Measure Enzymatic Activity in Reactions That Generate Inorganic Pyrophosphate

Cited by 83 publications

References 2 publications

Kinetic Evidence Is Consistent with the Rocker-Switch Mechanism of Membrane Transport by GlpT

Kinetic Evidence Is Consistent with the Rocker-Switch Mechanism of Membrane Transport by GlpT

Genomic identification and in vitro reconstitution of a complete biosynthetic pathway for the osmolyte di- myo -inositol-phosphate

The Escherichia coli fadK (ydiD) Gene Encodes an Anerobically Regulated Short Chain Acyl-CoA Synthetase

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