Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
2001-11-14
2004-06-08
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S194000, C435S007600, C435S007900
Reexamination Certificate
active
06746849
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to assays for nucleoside diphosphates, particularly ADP and GDP, and assays for nucleoside triphosphates, particularly ATP and GTP.
BACKGROUND ART
Nucleoside diphosphates and triphosphates play important roles in biology. ADP is the immediate precursor for the formation of ATP, the universal currency of cellular energy. GDP is a substrate for succinyl CoA synthetase, a key enzyme of the Krebs cycle, and is formed during gluconeogenesis by phosphoenolpyruvate carboxykinase. It is also essential in G-protein signalling, microtubule growth, and visual excitation. UDP is involved in the epimerisation of galactose to glucose, the formation of sucrose, and in the growth of glycogen. CDP is an important group in the synthesis of phosphoglycerides. Nucleoside diphosphates are also the products of reactions catalysed by several major classes of enzymes, such as triphosphatases and kinases, and are therefore produced by many cellular processes, including motility, muscle contraction, DNA synthesis, transcription, translation and nitrogen fixation.
The detection and measurement of nucleoside diphosphates and triphosphates is thus important in the study of biology and metabolism, particularly in bioenergetics.
Assays for ADP and ATP in biological samples based on luciferase have been known for over 20 years [e.g. refs 1, 2, 3, 4]. Bioluminescent assays for ADP and ATP have been described for use in muscle and adipose tissue biopsies [5] and a three-enzyme bioluminescent system utilising luciferase has been reported for use in bacterial cell extracts [6]. A bioluminescent ADP assay optimised for use at high ATP:ADP ratios has been reported [7], but this requires the enzymatic removal of ATP. In general, it is easier to measure ATP in the presence of ADP than to measure ADP in the presence of ATP.
Enzymatic spectrophotometric assays have also been described [e.g. 8].
Assays for GDP and GTP in biological samples are also well known [e.g., refs 9 & 10].
Reference 11 discloses column-based chromatographic assays for GDP, CDP and UDP. Radioactive assays for GDP and GTP have also been described [12, 13]. NMR-based assays for measuring in vivo ADP levels are known for yeast [14], and NMR has also been used to measure ADP and ATP and erythrocytes [15].
DISCLOSURE OF THE INVENTION
According to the present invention, nucleoside diphosphates are detected or measured by following the dephosphorylation of the phosphoenzyme form of nucleoside diphosphate kinase (NDPK), and nucleoside triphosphates are detected or measured by following the phosphorylation of NDPK to its phosphoenzyme form.
The invention thus provides (a) a process for detecting the presence of a nucleoside diphosphate in a sample, comprising the step of detecting the dephosphorylation of the phosphoenzyme form of a nucleoside diphosphate kinase, and (b) a process for detecting the presence of a nucleoside triphosphate in a sample, comprising the step of detecting the phosphorylation a nucleoside diphosphate kinase to the phosphoenzyme form.
The process will typically comprise the steps of:
causing nucleoside diphosphate in sample to bind to NDPK phosphoenzyme, or causing nucleoside triphosphate in sample to phosphorylate NDPK; and
detecting a change in a characteristic of the enzyme which differs between its phosphorylated and unphosphorylated forms.
The term “NDPK” means an enzyme having the activity of the enzyme as EC 2.7.4.6, namely the transfer of the &ggr;-phosphate group of a nucleoside triphosphate (N
1
TP) to a nucleoside diphosphate (N
2
DP) via a pin-pong mechanism:
N
1
TP+N
2
DP→N
1
DP+N
2
TP
Based on this reaction scheme, the systematic name of NDPK is “ATP:nucleoside-diphosphate phosphotransferase”, but the common name is “nucleoside diphosphate kinase”. The enzyme has also been variously described as kinase (phosphorylating), nucleoside diphosphate; nucleoside 5′-diphosphate kinase: nucleoside diphosphate (UDP) kinase: nucleoside diphosphokinase: nucleotide phosphate kinase: NM23.
NDPKs have been described for a number of organisms, both prokaryotic and eukaryotic e.g. human, cows, monkeys, mice. Xenopus, oats, peas, potatoes, yeast,
Bacillus subtilis, E. coli, Myxococcus xanthus,
avian myeloblastosis virus etc. These differ by cellular location, molecular weight, oligomeric structure, isoelectric point, reaction kinetics, substrate preference, pH optimum, pH range, temperature optimum, cation requirements (Mn
2+
, Mg
2−
, Co
2+
, Ca
2
etc.), and various isoforms have been described. Given the variety of suitable enzymes available, the skilled person can easily select and purify a NDPK to suit any particular situation.
The NDPK enzyme uses a ping-pong mechanism, transferring the &ggr;-phosphate from a nucleoside triphosphate (N
1
TP) to an active site histidine to form a phosphoenzyme intermediate, and then to a nucleoside diphosphate (N
2
DP). The invention is based on the finding that the phosphoenzyme intermediate is stable over a time-scale that allows its detection and measurement. Other enzymes that phosphorylate nucleoside diphosphates via a phosphoenzyme intermediate, preferably with a single binding site for nucleotide, may also be used in the invention.
The phosphoenzyme is able to transfer its phosphate group to N
2
DP in a sample to form the corresponding N
2
TP. Detection of this transfer can therefore be used for the detection of nucleoside diphosphate. To detect nucleoside diphosphate according to the invention, therefore, phosphoenzyme is required as a reagent. This can be readily formed by, for example, incubating NDPK with excess NTP, typically ATP. Formation of phosphoenzyme in this way is facilitated by removing Mg
2+
[16], for instance by using EDTA. Chemical phosphorylation of histidine using phosphormamide as a phosphorylating agent may also be used [17].
The phosphoenzyme can be isolated for use as a reagent. It has been found that the phosphoenzyme can be stored on ice for over 48 hours without dephosphorylation, and can be stored for longer periods (at least 5 months) at −80° C. (although repeated freeze-thawing results in some dephosphorylation). The stability of the phosphoenzyme over the time range needed for its preparation, and subsequently for monitoring kinetic events such as the release of ADP from an ATPase, is particularly advantageous.
When added to a sample containing NDP, the phosphoenzyme is dephosphorylated by the transfer of its phosphate group to the NDP. When added to a sample containing NTP, the phosphoenzyme is formed by the transfer of the NTP &ggr;-phosphate group to the enzyme. The invention relies on the ability to distinguish between the phosphorylated and dephosphorylated forms of NDPK.
In order to distinguish the phosphorylated and unphosphorylated forms of NDPK, any suitable measurable change can be used.
For instance, intrinsic properties of the enzyme can be used. Depending on the particular NDPK chosen, the following methods are examples of how dephosphorylation/phosphorylation may be detected, with varying levels of sensitivity:
The location of a phosphate (i.e. either bound to NDPK, or as the &ggr;-phosphate of a NTP) can be ascertained by following the
31
P NMR spectrum.
Protons whose environment changes upon dephosphorylation can be detected by, for instance NMR.
Dephosphorylation may cause a change in the fluorescence of a tryptophan residue in the protein [e.g., ref 18].
Dephosphorylation can be detected by following the loss of
32
P from radio-labelled phosphoenzyme. The radio-isotope can be conveniently incorporated into NDPK by using [&ggr;-
32
P]ATP.
Circular dichroism, or any other suitable spectrometric technique, can detect conformational changes which occur on dephosphorylation.
Dephosphorylation may result in a change in surface plasmon resonance properties.
Rather than using properties inherent in the wild-type enzyme, it may
Brune Martin Hermann Klemens
Corrie John Edgar Thomas
Webb Martin Ronald
Medical Research Council
Prouty Rebecca E.
Steadman David
Wenderoth , Lind & Ponack, L.L.P.
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