Inconsistencies between human genetic cytolocations and those derived using genomic sequence.

One result of the publishing of the human genome sequence is the ability to define objects through their position on the consensus sequence. While this has simplified the process of creating order maps for genes on a chromosome, it has created discrepancies between the published cytolocations of human genes, as presented through genetic references, and those locations derived computationally from the genomic sequence. For the 6,830 records with HUGO gene symbols shared between the online version of Mendelian Inheritance in Man and Ensembl, 18% of the records have a discrepancy of at least one cytogenetic band between the datasets. Discordance between data sets at this frequency would have a significant impact on the utility of datasets created by the amalgamation of numerous biological databases.

Painless electrodiagnostic current perception threshold and pain tolerance threshold values in CRPS subjects and healthy controls: a multicenter study.

The purpose of this study is to evaluate both painless and painful sensory transmission in patients with Complex Regional Pain Syndrome (CRPS) using the automated electrodiagnostic sensory Nerve Conduction Threshold (sNCT) test. This test generates reliable, painless Current Perception Threshold (CPT) and atraumatic Pain Tolerance Threshold (PTT) measures. Standardized CPT and PTT measures using constant alternating current sinusoid waveform stimulus at 3 different frequencies 5 Hz, 250 Hz, and 2 kHz (Neurometer CPT/C Neurotron, Inc. Baltimore, MD) were obtained from CRPS subjects at a distal phalange of the affected extremity and at an ipsilateral asymptomatic control site. Matched sites were tested on healthy subjects. Detection sensitivities for an abnormal PTT and CPT test were calculated based on specificity of 90% as determined from data obtained from healthy controls. A Spearman rank correlation was used to test for a significant association between presence of allodynia and an abnormal PTT or CPT at any frequency tested. Thirty-six CRPS subjects and 57 healthy controls were tested. The highest detection sensitivity of the PTT test from symptomatic test sites was 63% for the finger and 71% for the toe. PTT abnormalities were also detected, to a lesser degree, at the asymptomatic control site (41% finger control site, 16% toe control site). The highest CPT detection sensitivity at the symptomatic site was 37% for the finger site and 53% for the toe site. CPT abnormalities were also detected at the asymptomatic control site (29% finger control site, 37% toe control site). Eighty-six percent of the CRPS subjects had either a PTT or CPT abnormality at any frequency at the symptomatic site. There was a significant correlation between presence of allodynia and presence of an abnormal CPT and PTT, respectively (P < .01). The correlation coefficient was lower for CPT than for PTT, ie, 0.34 versus 0.6 for the finger and 0.48 versus 0.67 for the toe, respectively. In studied CRPS patients an abnormal PTT was detected with higher sensitivity than an abnormal CPT. Assessing PTT may become a useful electrodiagnostic quantitative sensory test for diagnosing and following the course of neuropathic pain conditions.

Preferential cerebrospinal fluid acetylcholinesterase inhibition by rivastigmine in humans.

This study sought to examine the feasibility of prolonged assessment of acetylcholinesterase (AChE) activity in the cerebrospinal fluid (CSF) of volunteers and to test the hypothesis that rivastigmine (ENA-713; Exelon, Novartis Pharma AG, Basel, Switzerland) selectively inhibits AChE in CSF in humans at a dose producing minimal inhibition of the peripheral enzyme. Lumbar CSF samples were collected continuously (0.1 mL x min(-1)) for 49 hours from eight healthy volunteers who took either placebo or a single oral dose of rivastigmine (3 mg). CSF specimens and samples of blood cells and blood plasma were analyzed at intervals for rivastigmine and its metabolite NAP 226-90 ([-] [3-([1-dimethylaminolethyl)-phenol]), erythrocyte AChE activity, CSF AChE activity, and plasma and CSF butyrylcholinesterase (BuChE) activity. Safety evaluations were performed 23 hours after drug dosing and at the end of the study. Evaluable data were obtained from six subjects. The mean time to maximal rivastigmine plasma concentration (tmax) was 0.83 +/- 0.26 hours, the mean maximal plasma concentration (Cmax) was 4.88 +/- 3.82 ng x mL(-1), the mean plasma area under the concentration versus time curve (AUC0-infinity) was 7.43 +/- 4.74 ng x hr x mL(-1), and the mean plasma t1/2 was 0.85 +/- 0.115 hours. The concentration of rivastigmine in CSF was lower than the quantification limit for assay (0.65 ng x mL(-1)), but NAP 226-90 reached a mean Cmax of 3.14 +/- 0.57 ng x mL(-1). Only minimal inhibition of erythrocyte AChE activity (approximately 3%) was observed. Inhibition of AChE in the CSF after rivastigmine administration was significantly greater than after placebo for up to 8.4 hours after the dose and was maximal (40%) at 2.4 hours. Plasma BuChE activity was significantly lower after rivastigmine than after placebo, but this was not clinically relevant. BuChE activity in CSF was significantly lower after rivastigmine than after placebo for up to 3.6 hours after dosing, but this difference was not sustained. This study confirms the feasibility of using continuous measurement of AChE activity in CSF over prolonged periods, that rivastigmine markedly inhibits CSF AChE after a single oral dose of 3 mg, and that the inhibition of central AChE is substantially greater than that of peripheral AChE or BuChE.

Long-term administration of controlled-release oxycodone tablets for the treatment of cancer pain.

We conducted a study of the safety of controlled-release (CR) oxycodone tablets (OxyContin Tablets) administered chronically to patients with cancer-related pain in a usual clinical setting. These patients had participated in 1 of 2 double-blind, active-control studies. Our study was an open, 3-month treatment study that included 87 patients. Patients received CR oxycodone tablets every 12 hr in a manner that reflected typical clinical practice. Supplemental immediate-release (IR) oxycodone was available PRN for breakthrough pain. Patients recorded medication use, adverse events, and evaluations of pain intensity and acceptability of therapy in a daily diary. Forty-four patients (51%) completed all 12 weeks of study; 43 patients (49%) discontinued participation. At baseline and throughout the study period, the overall mean pain-intensity score was slight to moderate. A comparison of initial and final doses showed a significant but modest increase in total daily CR oxycodone dose. An increase or decrease in titration of the oxycodone dose occurred for 66 patients (84%) at least once during the 12-week study period, primarily for increased pain. Forty-four patients (56%) did not undergo dose titration when the latter was indicated. Half of the patients used IR oxycodone rescue almost daily; the mean number of rescue doses per day was 1.5. Despite stable pain control and an increasing total daily CR oxycodone dose, the percentage of patients reporting common opioid-related adverse events decreased over the course of the study. CR oxycodone tablets administered every 12 hr were successfully used to manage cancer pain over a 12-week period. Importantly, side effects diminished over time without a concomitant change in efficacy.

The use of controlled-release oxycodone for the treatment of chronic cancer pain: a randomized, double-blind study.

To compare the effectiveness and safety of controlled-release (CR) oxycodone tablets with immediate-release (IR) oxycodone in patients with chronic cancer pain, a multicenter, randomized, double-blind, parallel-group study was performed in 111 patients with cancer pain. Patients were treated with 6 to 12 tablets or capsules of fixed-combination opioid/nonopioid analgesics per day at study entry. Patients received 30 mg of CR oxycodone tablets every 12 hr or 15 mg of IR oxycodone four times daily for 5 days. No titration or supplemental analgesic medications were permitted. The mean (+/- SE) baseline pain intensity (0 = none, 1 = slight, 2 = moderate, 3 = severe) was 1.5 +/- 0.1 for the CR oxycodone-treated group and 1.3 +/- 0.1 for the group given IR oxycodone (P > 0.05). The 5-day mean pain intensity was 1.4 +/- 0.1 and 1.1 +/- 0.1 for the CR and IR groups, respectively (P > 0.05). Discontinuation rates were equivalent (33%). There was no significant difference between treatment groups in the incidence of adverse events. This study demonstrates that cancer pain patients given 6 to 12 tablets or capsules of fixed-dose combination analgesics can be equally well treated with CR oxycodone administered every 12 hr or IR oxycodone four times daily at the same total daily dose. CR oxycodone offers the benefits of twice daily dosing.

Comparison of controlled-release and immediate-release oxycodone tablets in patients with cancer pain.

PURPOSE: This study compared the clinical efficacy of oxycodone hydrochloride controlled-release (CR) tablets administered every 12 hours with immediate-release (IR) oxycodone tablets administered four times daily in patients with cancer-related pain. PATIENTS AND METHODS: Cancer patients who required therapy for moderate to severe pain were randomized to CR oxycodone every 12 hours (n=81) or IR oxycodone four times daily (n=83) for 5 days in a multicenter, double-blind study. Pain intensity was assessed four times daily (categorical scale of none, slight, moderate, and severe); acceptability of therapy was assessed twice daily (categorical scale of very poor, poor, fair, good, and excellent). RESULTS: Pain intensity remained slight during the study, with mean oxycodone doses of 114 mg/d (range, 20 to 400 mg/d) for CR and 127 mg/d (range, 40 to 640 mg/d) for IR. Acceptability of therapy was fair to good with both treatments. While standard conversion ratios provided an acceptable dose for many patients, a protocol amendment that allowed initial titration and use of rescue medication reduced the discontinuation rate for lack of acceptable pain control (from 34% to 4% with CR and from 31% to 19% with IR before and after amendment, respectively) without increasing the discontinuation rate for adverse events (from 8% to 7% with CR and from 13% to 11% with IR). Fewer adverse events were reported with CR (109) than with IR (186) oxycodone (P=.006). CONCLUSION: CR oxycodone every 12 hours was as effective as IR oxycodone four times daily in managing moderate to severe cancer-related pain and was associated with fewer reports of adverse events.

High affinity binding of the pleckstrin homology domain of mSos1 to phosphatidylinositol (4,5)-bisphosphate.

mSos1 has been implicated in coupling mammalian tyrosine kinases to the Ras GTPase. Because activation of Ras induced by growth factor stimulation likely requires the localization of mSos1 to the plasma membrane, we have investigated the possibility that the PH domain of mSos1 might mediate an interaction of mSos1 with phospholipid membranes. A glutathione S-transferase fusion protein containing the pleckstrin homology (PH) domain of mSos1 bound specifically and tightly to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) with a Kd of 1.8 +/- 0.4 microM. This interaction was saturable and was competed away with the soluble head group of PI(4,5)P2, inositol 1,4, 5-triphosphate. Substitution of Arg452 within the PH domain with Ala had only a slight effect on binding to PI(4,5)P2, whereas substitution of Arg459 severely compromised the ability of the mSos1 PH domain to bind to PI(4,5)P2 containing vesicles. Purified full-length mSos1 and mSos1 complexed with Grb2 were also tested for binding to various phosphoinositol derivatives and demonstrated a specific interaction with PI(4,5)P2, although these interactions were weaker (Kd = approximately 53 and approximately 69 microM, respectively) than that of the PH domain alone. These findings suggest that the PH domain of mSos1 can interact in vitro with phospholipid vesicles containing PI(4,5)P2 and that this interaction is facilitated by the ionic interaction of Arg459 with the negatively charged head group of PI(4,5)P2. The association of the mSos1 PH domain with phospholipid may therefore play a role in regulating the function of this enzyme in vivo.

Do triage nurse-initiated X-rays for limb injuries reduce patient transit time?

Patients with isolated limb injuries are often required to wait a long time for treatment and investigation in emergency departments. It was hypothesized that allowing triage nurses to initiate X-rays would reduce transit times for these patients. A prospective, randomized comparison trial of 175 patients was conducted, comparing transit times between a group of patients who had X-rays initiated at triage and a group which did not. No statistically significant reduction in transit time was demonstrated by this change in practice, either for a group who had sustained fractures or for one which had not. Despite this finding, staff and patient satisfaction with this change in procedure was high. This justifies continuation of the practice and further research.

Characterization of the phosphotyrosine-binding domain of the Drosophila Shc protein.

The phosphotyrosine-binding (PTB) domain of Drosophila Shc (dShc) binds in vitro to phosphopeptides containing the sequence motif NPXpY, and physically associates with the activated Drosophila epidermal growth factor receptor homologue (DER) in vivo. The structural elements, specificity and binding kinetics of the dShc PTB domain have now been characterized. The dShc PTB domain appeared similar to the insulin-like receptor substrate-1 PTB domain in secondary structure as suggested by Fourier transform infrared spectroscopy. Surface plasmon resonance measurements indicated that the dShc PTB domain bound with high affinity to phosphopeptides (Der) derived from the Tyr1228 site of the DER receptor. The kinetics of the dShc PTB domain-Der phosphopeptide interaction differed from those of a typical SH2 domain-ligand interaction, in that the PTB domain displayed slower on/off rates. Competition binding assays using truncated versions of the Der peptides revealed that high affinity binding to the dShc PTB domain requires, in addition to the NPXpY motif, the presence of hydrophobic residues at both positions -5 and -7 relative to phosphotyrosine. The dShc PTB domain showed a similar binding specificity to the human Shc (hShc) PTB domain, but subtle differences were noted; such that the hShc PTB domain bound preferentially to a phosphopeptide from the mammalian nerve growth factor receptor, whereas the dShc PTB domain bound preferentially to phosphopeptides from the Drosophila DER receptor. The invertebrate dShc PTB domain therefore possesses a binding specificity for tyrosine-phosphorylated peptides that is optimally suited for recognition of the activated DER receptor.

Selective methyl group protonation of perdeuterated proteins.

Deuteration of aliphatic sites in proteins has shown great potential to increase the range of molecules amenable to study by NMR spectroscopy. One problem inherent in high-level deuterium incorporation is the loss of 1H-1H distance information obtainable from NOESY spectra of the labeled proteins. In the limit of perdeuteration, the available NH-NH NOEs are insufficient in many cases to define the three-dimensional structure of a folded protein. We describe here a method of producing proteins that retains all the advantages of perdeuteration, while enabling observation of many NOEs absent from spectra of fully deuterated samples. Overexpression of proteins in bacteria grown in 2H2O medium containing protonated pyruvate as the sole carbon source results in complete deuteration at C alpha and > 80% deuteration at C beta positions of nearly all amino acids. In contrast, the methyl groups of Ala, Val, Leu and Ile (gamma 2 only) remain highly protonated. This labeling pattern can be readily understood from analysis of bacterial pathways for pyruvate utilization and amino acid biosynthesis. As Ala, Val, Leu and Ile are among the most highly represented residue types in protein hydrophobic cores and at protein-protein interfaces, selectively methyl-protonated samples will be useful in many areas of structural analysis of larger molecules and molecular complexes by NMR.

Intrathecal ketorolac tromethamine produces analgesia after chronic constriction injury of sciatic nerve in rat.

PURPOSE: The study compared analgesic efficacy of intrathecally administered ketorolac tromethamine (K) and morphine hydrochloride (M) (in equimolar doses) in the chronic neuropathic pain model, induced by chronic constriction injury (CCI) of the sciatic nerve in rat. METHODS: Male Sprague-Dawley rats (n = 30) were anaesthetized with halothane and an intrathecal catheter was inserted to the mid-lumbar level of the spinal cord. On the 5th post-operative day, rats were anaesthetized with halothane and four ligatures were loosely applied around the right sciatic nerve. Seven days later, those animals were randomly divided into three groups and were injected with either saline, M (20 nmoles) or K (20 nmoles). Two pain responses (foot-withdrawal delay and hind paw elevation time) were measured on both sides using the radiant heat method. Further, thermal ("cold") allodynia was assessed by measuring of the total time of hind paw elevation in animals placed on the cold metal plate. RESULTS: Twenty nmoles of M and K injected intrathecally produced decrease of differential pain score calculated for both measured responses (hind paw withdrawal and hind paw elevation), compared with saline injected animals (P < 0.05). The reduction in pain response produced by K was less (P < 0.05). than the reduction in pain response observed in the animals receiving intrathecal M. Measurement of cold allodynia revealed that the animals in M and K injected groups demonstrated decreases in the total hind paw elevation time, when compared with saline-injected animals (P < 0.05). CONCLUSION: M and K produced hypoalgesia after intrathecal administration in rats with CCI, with M being more potent than K at an equimolar dose range. The analgesic effect of K was equal to equimolar doses of M for alleviation of cold allodynia.

Persistent pain associated with long-term intrathecal morphine administration.

A 32-year-old man with chronic intractable right lower extremity pain unresponsive to multiple neurosurgical and pharmacologic treatments, including intrathecal morphine administration, was successfully treated with sciatic nerve block, discontinuance of opioid therapy, and psychologic interventions. Plasma and urine ratios of morphine metabolites morphine-3-glucuronide and morphine-6-glucuronide were analyzed at the beginning of our interventions, and the results indicated that morphine-3-glucuronide levels were significantly higher than morphine-6-glucuronide levels. The possible association between the observed morphine metabolite ratio and the intractable pain in patients resistant to opioids may have potential clinical implications.

The in vitro translocase activity of lambda terminase and its subunits. Kinetic and biochemical analysis.

The terminase holoenzyme of bacteriophage lambda is a multifunctional protein composed of two subunits, gpNu1 and gpA. In vitro, under certain conditions, terminase can render DNAs from various sources, of varying lengths and termini, resistant to degradation by high concentrations of DNase I. This reaction is completely dependent on the presence of terminase, proheads, a hydrolyzable triphosphate, and a divalent metal ion, and we propose that it is the result of translocation of DNA into proheads by terminase. This reaction is stoichiometric with respect to terminase, DNA, and proheads and can be supported by all deoxyribo- and ribonucleoside triphosphates, but not by the corresponding diphosphates or nonhydrolyzable ATP analogs. Mg2+ and Ca2+ promote the reaction, but Mn2+ and Zn2+ do not. In the absence of spermidine, translocase activity is low, but addition of the Escherichia coli protein integration host factor (IHF) promotes specific translocation of only those DNA fragments containing the terminase-binding site, cosB. When spermidine is present, nonspecific translocation of DNA from any source is stimulated. Under these conditions IHF no longer promotes specificity, but translocation of only cosB-containing DNA fragments can be restored by addition of small amounts of a dialyzed and RNase-treated E. coli extract, suggesting that additional host factor(s) may be involved in determination of packaging specificity. To a limited extent, gpA alone can promote translocation, but gpNu1, which has no translocase activity on its own, must be added to approach the holoenzyme-like activity levels. Formation of viable phage cannot be accomplished by gpA in the absence of gpNu1.

A new procedure for the purification of the bacteriophage lambda terminase enzyme and its subunits. Properties of gene product A, the large subunit.

New methods for the purification of highly active bacteriophage lambda terminase holoenzyme, and its individual subunits, gene products (gp) A and gpNu1, have been developed. These methods are rapid, simple, reproducible, and give high yields of unaggregated protein from small volumes of culture. The procedures involve fractionation of extracts of Escherichia coli strains harboring plasmids engineered to overproduce the respective proteins. All purified proteins exist as monomers or dimers at moderate concentrations. At concentrations where holoenzyme efficiently promotes in vitro cosN-cleavage and lambda DNA packaging, gpA displays neither of these activities unless supplemented with gpNu1 and the E. coli protein integration host factor. At high protein concentrations, however, gpA can promote cos-cleavage by itself. Although gpNu1 itself cannot promote either cosN-cleavage or DNA packaging, it does modulate these activities of gpA. GpA is a DNA-stimulated ATPase whose catalytic parameters closely resemble those of the holoenzyme. Like the holoenzyme, gpA displays a DNA helicase activity which is able to melt the annealed cosN overhangs. Certain preparations of gpA appear to undergo a time-dependent amino-terminal clipping at discrete sites even in the presence of as many as four protease inhibitors and at low temperature.

The in vitro endonuclease activity of gene product A, the large subunit of the bacteriophage lambda terminase, and its relationship to the endonuclease activity of the holoenzyme.

The reaction requirements and kinetic properties of the in vitro endonuclease activity of the bacteriophage lambda terminase and its large subunit, gene product (gp) A, have been analyzed. Optimal cleavage reaction activity for both proteins requires Mg2+, a pH between 8.5 and 9.0, and is enhanced by ATP or ATP analogs. Under these conditions both terminase and gpA generate aberrant nicks in and around cosN. Optimal nicking specificity of terminase is observed under conditions of 50-100 mM salt, 5 mM spermidine, 1.5 mM ATP, and a pH between 7.0 and 7.5. Specific activity of terminase is greatly reduced under these conditions, and gpA is completely inactive at all protein concentrations tested. Under optimal reaction conditions, gpA endonuclease activity differs from that of the holoenzyme in that it can only be detected at high concentrations, is strongly protein concentration-dependent, and can not be stimulated by the Escherichia coli protein integration host factor. Addition of purified gpNu1 partially, but not completely, minimized these differences, suggesting that the role of gpNu1 in the holoenzyme is to modulate the basal endonuclease of gpA.

The in vitro ATPases of bacteriophage lambda terminase and its large subunit, gene product A. The relationship with their DNA helicase and packaging activities.

The bacteriophage lambda terminase is composed of two subunits, gpNu1 and gpA. In vitro, the holoenzyme is a site-specific endonuclease, helicase, ATPase, and can package lambda DNA into proheads. gpA possesses ATPase and helicase activities which are similar to those of the holoenzyme. Both terminase and gpA can hydrolyze a wide range of deoxyribo- and ribonucleoside triphosphates to inorganic phosphate and the corresponding diphosphate. Nucleoside diphosphates are not substrates for either protein. ATPase of both proteins is stimulated by double-stranded DNA. The ATPase of gpA is protein concentration-dependent, while that of terminase is not. Helicase activity of both proteins is not concentration-dependent, and requires a hydrolyzable triphosphate. ATP, dATP, and GTP supported helicase activity, while adenosine 5'-(beta, gamma-methylene)triphosphate, adenosine 5'-3-O-(thio)triphosphate, ADP, CTP, and UTP did not. The kinetic parameters of ATPase and helicase activities were similar for both proteins, but packaging with terminase was optimal only at a significantly higher level of ATP. Packaging was detectable at significant levels with CTP and UTP, but not with GTP. Packaging also differed from ATPase and helicase in the utilization of divalent metal cations and susceptibility to various inhibitors.

Infrared laser diode irradiation has no behavioral or biochemical effect on pain in the sciatic nerve ligation-induced mononeuropathy in rat.

The aim of this study was to evaluate the effect of acute and repeated (5 days) treatment with various types of infrared (IR) diode lasers and probes (single- vs cluster-beam) on the pain response in rats with peripheral mononeuropathy produced by sciatic nerve ligation. Male Sprague-Dawley rats were anesthetized with sodium pentobarbital, and the mid-thigh was surgically exposed to reveal the sciatic nerve, around which four ligatures were loosely tied. On postoperative day 5, the skin over the sciatic nerve lesion was subjected to a 30-min daily local exposure from a 904-nm IR diode laser (700 Hz, average output power 10 mW) with a single-beam probe, a 830-nm IR diode laser (700 Hz) with either a single-beam (average output power 50 mW) or cluster-beam probe (average output power 15 mW), or placebo for 5 consecutive days. Two pain responses (foot-withdrawal time and the hind-paw elevation time) were measured on both sides using the radiant heat method on days 5 and 9. In addition, cold allodynia was measured on day 9 of treatment by placing the rats on a chilled metal plate (4 degrees C) and measuring the duration of elevation of either of the hind paws. On day 9, the animals were sacrificed for collection of the samples of brain and lumbar spinal cord for the determination of the tissue concentrations of dynorphin A1-8-like immunoactivity (DYN) using specific radioimmunoassay (RIA). The hind-paw withdrawal and elevation times on the right side in all groups subjected to the various methods of IR laser irradiation did not differ significantly as compared with the placebo-treated group when measured on days 5 and 9 after surgery. No statistically significant differences in withdrawal response and elevation time of the unaffected left hind paw were noted either. The measurement of cold allodynia similarly failed to reveal any effect in laser-treated groups versus placebo. The RIA analysis found that tissue concentrations of DYN were significantly elevated in the spinal cord ipsilaterally to the ligation side, as compared with the contralateral side, in all rats with sciatic nerve ligation. All modalities of IR diode laser treatment did not produce any significant difference in the brain and spinal cord level of DYN on postoperative day 9 in all treatment groups. It is concluded that repeated IR diode laser treatment did not reduce hyperalgesia induced by sciatic nerve ligation in rats.

Threonine 246 at the active site of the L-lactate dehydrogenase of Bacillus stearothermophilus is important for catalysis but not for substrate binding.

Threonine 246 is an active site residue that is conserved in all known L-lactate dehydrogenase (LDH; EC 1.1.1.27) sequences. In order to investigate the role of Thr246 in Bacillus stearothermophilus LDH, this residue was altered by site-directed mutagenesis to valine, alanine, leucine, and serine, respectively. The effects of these mutations, as observed in both steady-state and single-turnover kinetic measurements with different substrates, demonstrated the importance for catalysis of a hydroxyl group in the 246 amino acid residue. In contrast, no significant contribution of the OH group of Thr246 to productive pyruvate binding was observed. Instead, it is proposed that the role of Thr246 may be to facilitate hydride transfer from the nicotinamide ring of the NADH cofactor to the pyruvate carbonyl group.