Immuno-diagnosis of Mycobacterium tuberculosis in sputum, and reduction of timelines for its positive cultures to within 3 h by pathogen-specific thymidylate kinase expression assays.

BACKGROUND: Laboratory diagnosis of Tuberculosis (TB) is traditionally based on microscopy and or culture. Microscopy is however, only sensitive to a specified degree of bacillary load not present in HIV co-infected persons. Traditional cultures of Mycobacterium tuberculosis (M. tb), on the other hand, take weeks to read-thereby delaying the critical decision whether or not, to treat. Although nucleic acids amplification tests (NAATS) applied directly on sputum or cultures can increase the sensitivity for TB diagnosis among those with HIV co-infection as well as reduce time-lines for positive culture detection, they do not replace the need for smear microscopy and culture. We have previously proposed the M. tb DNA-synthetic enzyme thymidylate kinase (aka TMKmt) as an organism-specific growth and proliferation biomarker to reduce time-lines for detection of positive TB cultures. In this study, we explored the secretory levels of TMKmt in M. tb cultures and sputum, towards improving the overall laboratory diagnosis of TB. METHODS AND RESULTS: Modelling of TMKmt secretion in vitro was done by cloning, expressing and SDS-PAGE/MALDI-TOF detection of purified recombinant TMKmt in E. coli. TMKmt expression profiling in M. tb was done by qRT-PCR assay of related messenger ribonucleic acids (mRNA) and TMKmt antigen detection by Enzyme linked Immuno-absorbent Assay (EIA) among cultures of a pathogenic wild-type Ugandan strain (genotype 1) alongside the H37Rv laboratory strain. Owing to the high-load of pathogen in-culture, direct EIA on limiting dilutions of sputum were done to examine for assay sensitivity. A rise in TMKmt antigen levels was observed at 3 h post-innoculation among in vitro cultures of E. coli. The 1st of several cyclic spikes in TMKmt mRNA and antigen levels were detected at 2.5 h among in vitro cultures of the pathogenic wild-type Ugandan isolate alongside the laboratory M. tb strain. TMKmt antigen was detected up to between 1 × 10-4-1 × 10-5 (containing 10 and 1 CFUs/ml) dilutions of a microscopically designated 1+ (est. Acid Fast Bacillary load of 1 × 105) patient sample. CONCLUSIONS: Detection of TMKmt expressed mRNA and Ag offers us opportune for instant diagnosis of M. tb in sputum, and reduction of timelines for positive pathogen detection in cultures to within 3 h.

2-Fluoro-ATP as a versatile tool for 19F NMR-based activity screening.

19F NMR-based methods have found utility in activity-based screening assays. However, because enzymes catalyze a diverse set of reactions, a large variety of fluorinated substrates would need to be identified to target each one separately. We have developed a more streamlined approach that is applicable to many enzymes that utilize ATP as a substrate. In this method, a fluorine-containing ATP analogue, 2-fluoro-ATP, is used to monitor the reaction. Applications are described for nicotinamide adenine dinucleotide synthetase and 3-phosphoinositide dependent kinase-1. Fragment screening results for the latter indicate that this technique can identify compounds that inhibit as well as activate reactions. The present results, together with previous biochemical studies from other laboratories, have shown that 2-fluoro-ATP can serve as a substrate for nine enzymes that are representative of three of the six enzyme subclasses, namely the transferases, hydrolases, and ligases. This suggests that 2-fluoro-ATP is suitable as a universal tool for screening ATP-requiring enzymes. Importantly, 2-fluoro-ATP has been determined to be a valid substrate for a variety of kinases, including both small molecule and protein kinases, suggesting that it may be useful for investigating the large number of pharmaceutically relevant kinases.

Differential proteome analysis of tonsils from children with chronic tonsillitis or with hyperplasia reveals disease-associated protein expression differences.

A proteomic approach has been used to establish a proteome map and differentiate between the protein composition of tonsils from patients with chronic tonsillitis (CT) and that of tonsils with hyperplasia (HPL). Two-dimensional gel analysis was performed with material from four patients with HPL and five patients with CT. An average of approximately 600 spots were detected in each gel. A total of 127 different proteins were identified in 158 spots analyzed by mass spectrometry. Our study revealed disease-associated differences between protein abundance for two protein spots, an HSP27 isoform and UMP-CMP kinase. Both protein spots were more abundant in the CT group. HSP27 ELISA was performed for 32 patients, 12 belonging to the HPL group and 20 to the CT group. ELISA could not be used to differentiate HSP27 isoforms nor to distinguish CT from HPL. HSP27 was found to migrate to two further protein spots in the 2D gels. The differently expressed HSP27 isoform migrated as the most acidic of all the HSP27 isoforms detected, indicating the highest degree of phosphorylation. The sum of all three HSP27 abundances in the gels from the CT group was not different from that of the HPL group, consistent with the ELISA results. Our results suggest that phosphorylation differences caused the observed migration differences of HSP27. Together with the UMP-CMP kinase abundance differences, we conclude that kinase and/or phosphatase activity are different in CT and HPL.

Human T-cell leukemia virus type 1 Tax oncoprotein induces and interacts with a multi-PDZ domain protein, MAGI-3.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL), whereas the closely related virus HTLV-2 has not been associated with such malignant conditions. HTLV-1 Tax1 oncoprotein transforms a rat fibroblast cell line (Rat-1) much more efficiently than does HTLV-2 Tax2. By using a differential display analysis, we isolated MAGI-3 as a Tax1-inducible gene in Rat-1 cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed that Tax1 induced MAGI-3 in Rat-1 cells. MAGI-3 has multiple PDZ domains and interacted with Tax1 but not Tax2 in 293T cells. The interaction of Tax1 with MAGI-3 was dependent on a PDZ domain-binding motif, which is missing in Tax2. The interaction of Tax1 with MAGI-3 altered their respective subcellular localization, and moreover, the interaction correlated well with the high transforming activities of Tax1 in Rat-1 cells relative to Tax2. MAGI-3 mRNA and the allied MAGI-1, but not MAGI-2, were expressed in HTLV-1-infected T-cell lines. Our results suggest that the interaction of Tax1 and MAGI-3 alters their respective biological activities, which may play a role in transformation by Tax1 as well as in the pathogenesis of HTLV-1-associated diseases.

Roles of immunoglobulin-like loops of junctional cell adhesion molecule 4; involvement in the subcellular localization and the cell adhesion.

BACKGROUND: Membrane-associated guanylate kinase with inverted domain structure-1 (MAGI-1) is a scaffolding protein at tight junctions (TJs). We have recently identified junctional adhesion molecule 4 (JAM4) as a MAGI-1-interacting protein. JAM4 belongs to the immunoglobulin superfamily and mediates Ca2+-independent adhesion. In this study, we examined the subcellular localization of JAM4 in various tissues and the involvement of JAM4 in the localization of MAGI-1. Moreover, we investigated into roles of immunoglobulin-like loops (Ig-loops) of JAM4. RESULTS: JAM4 was localized at TJs but also on apical membranes of epithelial cells in jejunum, ileum, and renal proximal tubules. In Madine Darby canine kidney (MDCK) cells, the localization of JAM4 at TJs depended on the first Ig-loop and did not require the MAGI-1-interacting region. JAM4 determined the subcellular localization of MAGI-1 in MDCK cells. In ileum, however, MAGI-1 was localized at TJs where JAM4 was not detected. Both of Ig-loops were necessary for homophilic interactions, but cis interactions depended on the first Ig-loop. CONCLUSION: JAM4 may be primarily targeted to apical membranes, and subsequently recruited to TJs through the first Ig-loop-mediated molecular interaction. JAM4 determines the localization of MAGI-1 in MDCK cells, but the in vivo localization of MAGI-1 does not necessarily depend on JAM4.

Selective reduction of a PDZ protein, SAP-97, in the prefrontal cortex of patients with chronic schizophrenia.

Many postsynaptic density proteins carrying postsynaptic density-95/discs large/zone occludens-1 (PDZ) domain(s) interact with glutamate receptors to control receptor dynamics and synaptic plasticity. Here we examined the expression of PDZ proteins, synapse-associated protein (SAP) 97, postsynaptic density (PSD)-95, chapsyn-110, GRIP1 and SAP102, in post-mortem brains of schizophrenic patients and control subjects, and evaluated their contribution to schizophrenic pathology. Among these PDZ proteins, SAP97 exhibited the most marked change: SAP97 protein levels were decreased to less than half that of the control levels specifically in the prefrontal cortex of schizophrenic patients. In parallel, its binding partner, GluR1, similarly decreased in the same brain region. The correlation between SAP97 and GluR1 levels in control subjects was, however, altered in schizophrenic patients. SAP102 levels were also significantly reduced in the hippocampus of schizophrenic patients, but this reduction was correlated with sample storage time and post-mortem interval. There were no changes in the levels of the other PDZ proteins in any of the regions examined. In addition, neuroleptic treatment failed to mimic the SAP97 change. These findings suggest that a phenotypic loss of SAP97 is associated with the postsynaptic impairment in prefrontal excitatory circuits of schizophrenic patients.

Blood-brain barrier tight junctions are altered during a 72-h exposure to lambda-carrageenan-induced inflammatory pain.

In this study, we examined the effect of lambda-carrageenan-induced inflammatory pain on the functional and structural properties of the rat blood-brain barrier (BBB) over a 72-h time period. Systemic inflammation was induced by an intraplantar injection of 3% lambda-carrageenan into the right hind paw of female Sprague-Dawley rats. In situ brain perfusion and Western blot analyses were performed at 1, 3, 6, 12, 24, 48, and 72 h. In situ brain perfusion showed lambda-carrageenan significantly increased brain uptake of [(14)C]sucrose at 1, 3, 6, and 48 h (139 +/- 9%, 166 +/- 19%, 138 +/- 13%, and 146 +/- 7% compared with control, respectively). Capillary depletion analysis insured the increased brain uptake was due to increased BBB permeability and not vascular trapping. Western blot analyses for zonula occludens-1 (ZO-1) and occludin were performed on isolated cerebral microvessels. ZO-1 expression was significantly increased at 1, 3, and 6 h and returned to control expression levels by 12 h. Total occludin expression was significantly reduced at 1, 3, 6, 12, and 48 h. This investigation demonstrated that lambda-carrageenan-induced inflammatory pain elicits a biphasic increase in BBB permeability with the first phase occurring from 1-6 h and the second phase occuring at 48 h. Furthermore, changes in BBB function are correlated with altered tight junctional protein expression of occludin and ZO-1. Changes in the structure of tight junctions may have important clinical ramifications concerning central nervous system homeostasis and therapeutic drug delivery.

MAGI-1: a widely expressed, alternatively spliced tight junction protein.

Tight junctions are apically localized structures that regulate the passage of small molecules and proteins through intercellular regions of epithelial or endothelial cells. These structures are complex multimolecular assemblages that contain both transmembrane and membrane-associated proteins. MAGUKs (Membrane-Associated Guanylate Kinases) are a family of scaffolding proteins that contain multiple protein interaction domains, including PDZ, SH3, WW, and guanylate kinase motifs, and have been grouped into five discrete subfamilies based on homology. Little is known regarding the most recently described subfamily of MAGUKs, termed MAGIs (MAGUKS with Inverted domain structure). Here we show that two of the three known MAGI isoforms, MAGI-1 and MAGI-3, are present in the tight junctions of cultured epithelial cells. A broader examination of MAGI-1 expression in vivo shows that it is present in the tight junctions of all epithelial cell types examined. Human MAGI-1 transcripts are alternatively spliced at three sites, and two forms are expressed only in nonepithelial tissues, predominantly in brain. The major form that is expressed in cultured colon carcinoma epithelial cells, as well as several epithelial-rich tissues, contains an extended carboxy terminus encoding potential nuclear targeting signals. MAGI-1, ZO-1, and ZO-2 all colocalize in nonpolarized epithelial cells, suggesting that they form a preassembled complex that is incorporated into the tight junction upon polarization. Finally, all of the alternatively spliced forms of MAGI-1 show tight junction localization, and this localization occurs in the absence of the guanylate kinase and WW domains as well as the extended carboxy terminus.

Parkin and CASK/LIN-2 associate via a PDZ-mediated interaction and are co-localized in lipid rafts and postsynaptic densities in brain.

Mutations in the gene encoding parkin cause an autosomal recessive juvenile-onset form of Parkinson's disease. Parkin functions as a RING-type E3 ubiquitin-ligase, coordinating the transfer of ubiquitin to substrate proteins and thereby targeting them for degradation by the proteasome. We now report that the extreme C terminus of parkin, which is selectively truncated by a Parkinson's disease-causing mutation, functions as a class II PDZ-binding motif that binds CASK, the mammalian homolog of Caenorhabditis elegans Lin-2, but not other PDZ proteins in brain extracts. Importantly, parkin co-localizes with CASK at synapses in cultured cortical neurons as well as in postsynaptic densities and lipid rafts in brain. Further, parkin associates not only with CASK but also with other postsynaptic proteins in the N-methyl d-aspartate (NMDA) receptor-signaling complex, in rat brain in vivo. Finally, despite exhibiting E2-dependent ubiquitin ligase activity, rat brain parkin does not ubiquitinate CASK, suggesting that CASK may function in targeting or scaffolding parkin within the postsynaptic complex rather than as a direct substrate for parkin-mediated ubiquitination. These data implicate for the first time a PDZ-mediated interaction between parkin and CASK in neurodegeneration and possibly in ubiquitination of proteins involved in synaptic transmission and plasticity.

Electron microscopic immunocytochemical detection of PSD-95, PSD-93, SAP-102, and SAP-97 at postsynaptic, presynaptic, and nonsynaptic sites of adult and neonatal rat visual cortex.

Membrane-associated guanylate kinases (MAGUKs) assemble protein complexes at sites of cell-cell contact. At excitatory synapses in brain, MAGUKs localize to the postsynaptic density (PSD) and interact with N-methyl-D-aspartate (NMDA) glutamate receptors and downstream signaling proteins. However, NMDA receptors are not restricted to the PSDs, as electron microscopic immunocytochemical (EM-ICC) results indicate that NMDA receptors also occur at nonsynaptic portions of dendrites, perhaps functioning as reserves for rapid insertion into synaptic membranes in response to appropriate synaptic activity. NMDA receptors also occur in axons, at least in part to support glutamate-dependent enhancement of transmitter release. In this study, a systematic EM-ICC survey was performed to determine whether the distributions of four neuronal MAGUKs-PSD-95, PSD-93, SAP-102, and SAP-97-resemble that of NMDA receptors. Quantitative analysis revealed that the density of PSD-95 over thick PSDs of asymmetric axo-spinous synaptic junctions is 2-3-fold the level in the immediately adjacent cytoplasm of spines and terminals, while symmetric synapses show no association with PSD-95. Similarly, all four MAGUKs occur over PSDs of spines. However, we also detected MAGUK immunoreactivity, albeit more diffusely, along presynaptic membranes and in the cytoplasm of axons and dendritic shafts. In fact, the overall distribution of PSD-95 within the neuropil is equally prevalent along plasma membranes (including synaptic portions) as in the cytoplasm, away from plasma membranes. These results suggest that MAGUKs have dual roles: to maintain receptors at synapses and to regulate shuttling of receptors between nonsynaptic and synaptic sites.

Distribution of members of the PSD-95 family of MAGUK proteins at the synaptic region of inner and outer hair cells of the guinea pig cochlea.

PDZ-domain containing proteins of the MAGUK (membrane-associated guanylate kinase) family target, anchor, and cluster receptors and channels to subcellular sites. Among the MAGUK proteins, the members of the PSD-95 family (MAGUKs: PSD-95, PSD-93, SAP-97, and SAP-102) target and anchor glutamate receptors to the synaptic terminals. Associations of glutamate receptors with MAGUKs have been described in the brain but not in the cochlea. In this study, RT-PCR, immunofluorescence microscopy, and immunoelectron microscopy were used to investigate the presence and distribution of MAGUK proteins in the organ of Corti. The presence of the mRNA for PSD-95, PSD-93, SAP-97, and SAP-102 in the organ of Corti was confirmed by RT-PCR. Immunocytochemistry using a "pan-MAGUK" antibody, which recognizes all four MAGUK proteins, and selective antibodies against these proteins revealed that all four MAGUKs are present within the base of inner hair cells while all except SAP-97 are found within the base of the outer hair cells. In addition, PSD-93 and PSD-95 are found in postsynaptic afferent terminals on inner hair cells, while postsynaptic afferent terminals on outer hair cells have PSD-93.

The ligand affinity of proteins measured by isothermal denaturation kinetics.

An isothermal denaturation kinetic method was developed for identifying potential ligands of proteins and measuring their affinity. The method is suitable for finding ligands specific toward proteins of unknown function and for large-scale drug screening. It consists of analyzing the kinetics of isothermal denaturation of the protein-with and without the presence of potential specific ligands-as measured by long-wavelength fluorescent dyes whose quantum yield increases when bound to hydrophobic regions exposed upon unfolding of the proteins. The experimental procedure was developed using thymidylate kinase and stromelysin as target proteins. The kinetics of thermal unfolding of both of these enzymes were consistent with a pathway of two consecutive first-order rate-limiting steps. Reflecting the stabilizing effect of protein/ligand complexes, the presence of specific ligands decreased the value of the rate constants of both steps in a dose-dependent manner. The dependence of the rate constants on ligand concentration obeyed a simple binding isotherm, the analysis of which yielded an accurate equilibrium constant for ligand binding. The method was validated by comparing its results with those obtained under the same conditions by steady-state fluorescence spectroscopy, circular dichroism, and uv spectrophotometry: The corresponding rate constants were comparable for each of the analytical detection methods.

Fetal life in Down syndrome starts with normal neuronal density but impaired dendritic spines and synaptosomal structure.

Information on fetal brain in Down Syndrome (DS) is limited and there are only few histological, mainly anecdotal reports and no systematic study on the wiring of the brain in early prenatal life exists. Histological methods are also hampered by inherent problems of morphometry of neuronal structures. It was therefore the aim of the study to evaluate neuronal loss, synaptic structures and dendritic spines in the fetus with Down Syndrome as compared to controls by biochemical measurements. 2 dimensional electrophoresis with subsequent mass spectroscopical identification of spots and their quantification with specific software was selected. This technique identifies proteins unambiguously and concomitantly on the same gel. Fetal cortex samples were taken at autopsy with low post-mortem time, homogenized and neuron specific enolase (NSE) determined as a marker for neuronal density, the synaptosomal associated proteins alpha SNAP [soluble N-ethylmaleimide-sensitive fusion (NSF) attachment protein], beta SNAP, SNAP 25 and the channel associated protein of synapse 110 (chapsyn 110) as markers for synaptosomal structures and drebrin (DRB) as marker for dendritic spines. NSE, chapsyn 110 and beta SNAP were comparable in the control fetus panel and in Down Syndrome fetuses. Drebrin was significantly and remarkably reduced and not even detectable in several Down Syndrome brain samples. Quantification of SNAP 25 revealed significantly reduced values in DS cortex and alpha SNAP was only present in half of the DS individuals. We conclude that at the time point of about 19 weeks of gestation (early second trimester) no neuronal loss can be detected but drebrin, a marker for dendritic spines and synaptosomal associated proteins alpha SNAP and SNAP 25 were significantly reduced indicating impaired synaptogenesis. Early dendritic deterioration maybe leading to the degeneration of the dendritic tree and arborization, which is a hallmark of Down Syndrome from infancy.

Immunochemical analysis of UMP kinase from Escherichia coli.

Mono- and polyclonal antibodies directed against UMP kinase from Escherichia coli were tested with the intact protein or with fragments obtained by deletion mutagenesis. As detected in enzyme-linked immunosorbent assay tests, the carboxy-terminal quarter of UMP kinase is immunodominant. Polyclonal antibodies inhibited the enzyme activity with partial or total loss of allosteric effects exerted by UTP and GTP, respectively. These data indicate that the UTP and GTP binding sites in UMP kinase are only partially overlapping. One monoclonal antibody (44-2) recognized a linear epitope in UMP kinase between residues 171 and 180. A single substitution (D174N) in this segment of the enzyme abolished its interaction with the monoclonal antibody (44-2). Polyclonal antisera were used to identify UMP kinase in the bacterial proteome. The enzyme appears as a single spot on two-dimensional electrophoresis at a pI of 7.24 and an apparent molecular mass of 26 kDa. Immunogold labeling of UMP kinase in whole E. coli cells shows a localization of the protein near the bacterial membranes. Because the protein does not contain sequences usually required for compartmentalization, the aggregation properties of UMP kinase observed in vitro might play a role in this phenomenon. The specific localization of UMP kinase might also be related to its putative role in cell division.

Localization of membrane-associated guanylate kinase (MAGI)-1/BAI-associated protein (BAP) 1 at tight junctions of epithelial cells.

Membrane-associated guanylate kinase (MAGI)-1/BAI-associated protein (BAP) 1 and Synapse-associated protein (SAP) 97/human Discs-large tumor suppressor gene (hDLG) are ubiquitous isoforms of synaptic scaffolding molecule (S-SCAM) and Postsynaptic density (PSD)-95/SAP90, both of which are implicated in the structures of synapses, respectively. SAP97/hDLG is localized at epithelial junctions and may function as a scaffolding protein, but the subcellular localization or the function of MAGI-1/BAP1 has not been clarified. In intestinal epithelial cells, MAGI-1/BAP1 was localized at tight junctions, whereas SAP97/hDLG was localized diffusely at cell - cell junctions. In Madine Darby canine kidney (MDCK) cells, MAGI-1/BAP1 was colocalized with ZO-1, whereas SAP97/hDLG was colocalized with E-cadherin. In MDCK cells, dominant active and negative mutants of Rac1 small G protein changed the amounts of SAP97/hDLG at cell - cell junctions, but not that of MAGI-1/BAP1. When MDCK cells were switched to a low Ca2+ medium, E-cadherin disappeared from the plasma membrane, and cells were dissociated. The phorbol 12-myristate 13-acetate-treatment after the low Ca2+ switch induced a tight junction-like structure. MAGI-1/BAP1 was recruited with ZO-1 to this structure, but SAP97/hDLG or E-cadherin was not. These findings suggest that MAGI-1/BAP1 is a component of tight junctions of epithelial cells, and that its role is different from that of SAP97/hDLG.

A tripartite protein complex with the potential to couple synaptic vesicle exocytosis to cell adhesion in brain.

We identify a complex of three proteins in brain that has the potential to couple synaptic vesicle exocytosis to neuronal cell adhesion. The three proteins are: (1) CASK, a protein related to MAGUKs (membrane-associated guanylate kinases); (2) Mint1, a putative vesicular trafficking protein; and (3) Veli1, -2, and -3, vertebrate homologs of C. elegans LIN-7. CASK, Mint1, and Velis form a tight, salt-resistant complex that can be readily isolated. CASK, Mint1, and Velis contain PDZ domains in addition to other modules. However, no PDZ domains are involved in complex formation, leaving them free to recruit cell adhesion molecules, receptors, and channels to the complex. We propose that the tripartite complex acts as a nucleation site for the assembly of proteins involved in synaptic vesicle exocytosis and synaptic junctions.

[The effect of ionizing radiation and physical loading on the molecular heterogeneity of nucleoside phosphate kinases in liver subcellular fractions]. / Vliianie ioniziruiushchei radiatsii i fizicheskoi nagruzki na molekuliarnuiu geterogennost' nukleozidfosfatkinaz v subkletochnykh fraktsiiakh pecheni.

The different molecular forms of nucleoside-monophosphate kinases (KF 2.7.4.4) and nucleoside-diphosphate kinases (KF 1.7.4.6) which are responsible for the final steps of pyrimidine nucleotide synthesis were determined in mitochondrial rat hepatic supernatant under condition of combined influence of X-ray and maximum physical exercises (running up to complete exhaustion). The maximum activity of investigated nucleosidediphosphate kinases were observed in intact animals in that fractions which were eluted with tris-HCl buffer solution (0.075 and 1.0 M, pH 7.4). X-ray radiation and physical exercises caused the deviation of chromatographic data of maximal enzymatic activities. The drastic lowering of investigated enzymes was observed after X-ray irradiation and maximum physical exhaustion. This fact is in favour for the suppression of the final steps of primidine nucleotides synthesis under explored conditions of experimental investigations.

Bioluminescent determination of 0.1 picomole amounts of guanine nucleotides.

A bioluminescence procedure for the determination of the guanylates has been optimized to allow measurement of 0.1 pmol amounts. Modifications of the Karl procedure include the use of purified firefly luciferase and nucleoside diphosphate kinase instead of a crude extract of firefly tails, the use of Tricine buffer instead of the inhibitory arsenate buffer, and optimization of the amounts of reagents and incubation times for each of the partial reactions. In the determination of GMP, background values varied widely with different lots of bovine guanylate kinase. Careful selection of a suitable lot of bovine brain guanylate kinase was essential for determination of lower amounts of guanylates. This establishes that selection of guanylate kinase must be based on experimental determination and not reported adenylate kinase activity. The wide variation in background was not eliminated by the inclusion of adenylate kinase inhibitors.