TTX-sensitive and TTX-insensitive sodium channel mRNA transcripts are independently regulated in adult skeletal muscle after denervation.

The expression of mRNA encoding the TTX-sensitive (SkM1) and TTX-insensitive (SkM2) voltage-dependent sodium channels in adult skeletal muscle is independently regulated. In normal skeletal muscle, only the SkM1 message is expressed and the level varies with muscle fiber type. After surgical denervation, the steady-state SkM1 mRNA level declines transiently, but returns to control levels within 5 days. Expression of SkM2 transcripts is markedly activated, reaching a peak 3 days after axotomy and then declining to a maintained level at approximately 30% of peak. Chemical denervation with botulinum toxin results in higher levels of SkM2 mRNA, which by 7 days posttreatment are 7-fold greater than levels in paired axotomized muscles. SkM2 expression subsequently declines as functional reinnervation appears. Quantal acetylcholine release appears to play a major role in suppression of SkM2 expression in adult innervated or reinnervated muscle, whereas nonquantal factors in toxin-treated, but not axotomized, muscle may sustain high level SkM2 mRNA expression.

Primary structure and expression of a sodium channel characteristic of denervated and immature rat skeletal muscle.

The alpha subunit of a voltage-sensitive sodium channel characteristic of denervated rat skeletal muscle was cloned and characterized. The cDNA encodes a 2018 amino acid protein (SkM2) that is homologous to other recently cloned sodium channels, including a tetrodotoxin (TTX)-sensitive sodium channel from rat skeletal muscle (SkM1). The SkM2 protein is no more homologous to SkM1 than to the rat brain sodium channels and differs notably from SkM1 in having a longer cytoplasmic loop joining domains 1 and 2. Steady-state mRNA levels for SkM1 and SkM2 are regulated differently during development and following denervation: the SkM2 mRNA level is highest in early development, when TTX-insensitive channels predominate, but declines rapidly with age as SkM1 mRNA increases; SkM2 mRNA is not detectable in normally innervated adult skeletal muscle but increases greater than 100-fold after denervation; rat cardiac muscle has abundant SkM2 mRNA but no detectable SkM1 message. These findings suggest that SkM2 is a TTX-insensitive sodium channel expressed in both skeletal and cardiac muscle.

Primary structure and functional expression of a mammalian skeletal muscle sodium channel.

We describe the isolation and characterization of a cDNA encoding the alpha subunit of a new voltage-sensitive sodium channel, microI, from rat skeletal muscle. The 1840 amino acid microI peptide is homologous to alpha subunits from rat brain, but, like the protein from eel electroplax, lacks an extended (approximately 200) amino acid segment between homologous domains I and II. Northern blot analysis indicates that the 8.5 kb microI transcript is preferentially expressed in skeletal muscle. Sodium channels expressed in Xenopus oocytes from synthetic RNA encoding microI are blocked by tetrodotoxin and mu-conotoxin at concentrations near 5 nM. The expressed sodium channels have gating kinetics similar to the native channels in rat muscle fibers, except that inactivation occurs more slowly.

A carboxy-terminal alpha-helical segment in the rat skeletal muscle voltage-dependent Na+ channel is responsible for its interaction with the amino-terminus.

Cytoplasmic segments of the adult rat skeletal muscle sodium channel alpha-subunit (rSkM1) comprise a major portion (approximately 40%) of the total protein and are involved in channel functions both general, such as inactivation, and isoform-specific, for example, protein kinase A modulation. Far ultraviolet circular dichroism measurements of synthetic peptides and overexpressed fusion proteins containing individual channel cytoplasmic segments suggest that cytoplasmic domains of rSkM1 contain ordered secondary structures even in the absence of adjoining transmembrane segments. Intrinsic fluorescence experiments with a nested set of carboxy-terminal deletion proteins confirm a specific interaction between the channel's amino- and carboxy-termini and identify residues 1716-1737 in the carboxy-terminus as the region that binds to the amino-terminus. Circular dichroism measurements suggest that this same region is organized as an alpha-helix and that electrostatic forces may contribute to this association. The interaction of the amino- and carboxy-termini is not accompanied by secondary structure changes detectable by circular dichroism spectroscopy, but a decrease in intrinsic fluorescence indicates that this association is accompanied by a change in the environment of Trp1617.

Role of an S4-S5 linker in sodium channel inactivation probed by mutagenesis and a peptide blocker.

A pair of conserved methionine residues, located on the cytoplasmic linker between segments S4 and S5 in the fourth domain of human heart Na channels (hH1), plays a role in the kinetics and voltage dependence of inactivation. Substitution of these residues by either glutamine (M1651M1652/QQ) or alanine (MM/AA) increases the inactivation time constant (tau) at depolarized voltages, shifts steady-state inactivation (h infinity) in a depolarized direction, and decreases the time constant for recovery from inactivation. The data indicate that the mutations affect the rate constants for both binding and unbinding of a hypothetical inactivation particle from its binding site. Cytoplasmic application of the pentapeptide KIFMK in Na channels mutated to remove inactivation produces current decays resembling inactivation (Eaholtz, G., T. Scheuer, and W.A. Catterall. 1994. Neuron. 12: 1041-1048.). KIFMK produces a concentration-dependent, voltage-independent increase in the decay rate of MM/QQ and MM/AA currents at positive membrane potentials (Ki approximately 30 microM), while producing only a small increase in the decay rate of wild-type currents at a concentration of 200 microM. Although MM/QQ inactivates approximately 2.5-fold faster than MM/AA in the absence of peptide, the estimated rate constants for peptide block and unblock do not differ in these mutants. External Na+ ions antagonize the block by cytoplasmic KIFMK of MM/AA channels, but not the inactivation kinetics of this mutant in the absence of peptide. The effect of external [Na+] is interpreted as a voltage-dependent knock-off mechanism. The data provide evidence that KIFMK can only block channels when they are open and that peptide block does not mimic the inactivation process.

A unique role for the S4 segment of domain 4 in the inactivation of sodium channels.

Sodium channels have four homologous domains (D1-D4) each with six putative transmembrane segments (S1-S6). The highly charged S4 segments in each domain are postulated voltage sensors for gating. We made 15 charge-neutralizing or -reversing substitutions in the first or third basic residues (arginine or lysine) by replacement with histidine, glutamine, or glutamate in S4 segments of each domain of the human heart Na+ channel. Nine of the mutations cause shifts in the conductance-voltage (G-V) midpoints, and all but two significantly decrease the voltage dependence of peak Na+ current, consistent with a role of S4 segments in activation. The decreases in voltage dependence of activation were equivalent to a decrease in apparent gating charge of 0.5-2.1 elementary charges (eo) per channel for single charge-neutralizing mutations. Three charge-reversing mutations gave decreases of 1.2-1.9 eo per channel in voltage dependence of activation. The steady-state inactivation (h infinity) curves were fit by single-component Boltzmann functions and show significant decreases in slope for 9 of the 15 mutants and shifts of midpoints in 9 mutants. The voltage dependence of inactivation time constants is markedly decreased by mutations only in S4D4, providing further evidence that this segment plays a unique role in activation-inactivation coupling.

Evidence for a direct interaction between internal tetra-alkylammonium cations and the inactivation gate of cardiac sodium channels.

The effects of internal tetrabutylammonium (TBA) and tetrapentylammonium (TPeA) were studied on human cardiac sodium channels (hH1) expressed in a mammalian tsA201 cell line. Outward currents were measured at positive voltages using a reversed Na gradient. TBA and TPeA cause a concentration-dependent increase in the apparent rate of macroscopic Na current inactivation in response to step depolarizations. At TPeA concentrations < 50 microM the current decay is well fit by a single exponential over a wide voltage range. At higher concentrations a second exponential component is observed, with the fast component being dominant. The blocking and unblocking rate constants of TPeA were estimated from these data, using a three-state kinetic model, and were found to be voltage dependent. The apparent inhibition constant at 0 mV is 9.8 microM, and the blocking site is located 41 +/- 3% of the way into the membrane field from the cytoplasmic side of the channel. Raising the external Na concentration from 10 to 100 mM reduces the TPeA-modified inactivation rates, consistent with a mechanism in which external Na ions displace TPeA from its binding site within the pore. TBA (500 microM) and TPeA (20 microM) induce a use-dependent block of Na channels characterized by a progressive, reversible, decrease in current amplitude in response to trains of depolarizing pulses delivered at 1-s intervals. Tetrapropylammonium (TPrA), a related symmetrical tetra-alkylammonium (TAA), blocks Na currents but does not alter inactivation (O'Leary, M. E., and R. Horn. 1994. Journal of General Physiology. 104:507-522.) or show use dependence. Internal TPrA antagonizes both the TPeA-induced increase in the apparent inactivation rate and the use dependence, suggesting that all TAA compounds share a common binding site in the pore. A channel blocked by TBA or TPeA inactivates at nearly the normal rate, but recovers slowly from inactivation, suggesting that TBA or TPeA in the blocking site can interact directly with a cytoplasmic inactivation gate.

A molecular link between activation and inactivation of sodium channels.

A pair of tyrosine residues, located on the cytoplasmic linker between the third and fourth domains of human heart sodium channels, plays a critical role in the kinetics and voltage dependence of inactivation. Substitution of these residues by glutamine (Y1494Y1495/QQ), but not phenylalanine, nearly eliminates the voltage dependence of the inactivation time constant measured from the decay of macroscopic current after a depolarization. The voltage dependence of steady state inactivation and recovery from inactivation is also decreased in YY/QQ channels. A characteristic feature of the coupling between activation and inactivation in sodium channels is a delay in development of inactivation after a depolarization. Such a delay is seen in wild-type but is abbreviated in YY/QQ channels at -30 mV. The macroscopic kinetics of activation are faster and less voltage dependent in the mutant at voltages more negative than -20 mV. Deactivation kinetics, by contrast, are not significantly different between mutant and wild-type channels at voltages more negative than -70 mV. Single-channel measurements show that the latencies for a channel to open after a depolarization are shorter and less voltage dependent in YY/QQ than in wild-type channels; however the peak open probability is not significantly affected in YY/QQ channels. These data demonstrate that rate constants involved in both activation and inactivation are altered in YY/QQ channels. These tyrosines are required for a normal coupling between activation voltage sensors and the inactivation gate. This coupling insures that the macroscopic inactivation rate is slow at negative voltages and accelerated at more positive voltages. Disruption of the coupling in YY/QQ alters the microscopic rates of both activation and inactivation.

Glutamine substitution at alanine1649 in the S4-S5 cytoplasmic loop of domain 4 removes the voltage sensitivity of fast inactivation in the human heart sodium channel.

Normal activation-inactivation coupling in sodium channels insures that inactivation is slow at small but rapid at large depolarizations. M1651Q/M1652Q substitutions in the cytoplasmic loop connecting the fourth and fifth transmembrane segments of Domain 4 (S4-S5/D4) of the human heart sodium channel subtype 1 (hH1) affect the kinetics and voltage dependence of inactivation (Tang, L., R.G. Kallen, and R. Horn. 1996. J. Gen. Physiol. 108:89-104.). We now show that glutamine substitutions NH2-terminal to the methionines (L1646, L1647, F1648, A1649, L1650) also influence the kinetics and voltage dependence of inactivation compared with the wild-type channel. In contrast, mutations at the COOH-terminal end of the S4-S5/D4 segment (L1654, P1655, A1656) are without significant effect. Strikingly, the A1649Q mutation renders the current decay time constants virtually voltage independent and decreases the voltage dependences of steady state inactivation and the time constants for the recovery from inactivation. Single-channel measurements show that at negative voltages latency times to first opening are shorter and less voltage dependent in A1649Q than in wild-type channels; peak open probabilities are significantly smaller and the mean open times are shorter. This indicates that the rate constants for inactivation and, probably, activation are increased at negative voltages by the A1649Q mutation reminiscent of Y1494Q/ Y1495Q mutations in the cytoplasmic loop between the third and fourth domains (O'Leary, M.E., L.Q. Chen, R.G. Kallen, and R. Horn. 1995. J. Gen. Physiol. 106:641-658.). Other substitutions, A1649S and A1649V, decrease but fail to eliminate the voltage dependence of time constants for inactivation, suggesting that the decreased hydrophobicity of glutamine at either residues A1649 or Y1494Y1495 may disrupt a linkage between S4-S5/D4 and the interdomain 3-4 loop interfering with normal activation-inactivation coupling.

The characterization of atrial natriuretic peptide (ANP) expression by primary cultures of atrial myocytes using an ANP-specific monoclonal antibody and an ANP messenger ribonucleic acid probe.

The biochemical and morphological characteristics of primary neonatal rat atrial myocytes were examined in order to establish a model system for future studies of the biosynthesis and secretion of atrial natriuretic peptide (ANP). Preliminary studies demonstrated that the quantity of immunoactive ANP/microgram protein within rat atria increased as a function of age from 2 ng/micrograms in 19 day prenatal animals to 400 ng/micrograms in the adult. Gel filtration, reversed phase HPLC, and ion exchange HPLC indicated that there were similar quantities of immunoactive ANP in the right and left atria at various ages, and that the major molecular form of the peptide in the heart is chromatographically indistinguishable from ANP(1-126). Cultures of dissociated cells were prepared from pooled left and right atria derived from 1 day postnatal animals. A complete serum-free medium was developed which resulted in the maintenance of high levels of immunoactive ANP in the cultures. As determined by RIA, the cellular content of ANP increased in the cultures as a function of time through 7 days in vitro. The quantity of immunoactive ANP in the cultures increased approximately 2- to 3-fold between days 3 and 7. When the cultures that had been maintained for 7 days were submitted to immunocytochemistry using an ANP-specific monoclonal antibody, distinct colonies of spindle-shaped cells stained positively. In situ hybridization, utilizing an 35S-labeled ANP messenger RNA probe, demonstrated that these colonies of myocytes expressed the ANP message. Using quantitative dot-blot hybridization it was shown that the ANP mRNA level increased approximately 50-fold between days 1 and 7 in culture. These studies indicate that the serum-free culture medium allows continued accumulation of both ANP and the ANP message in culture and will provide a useful model system to characterize factors that regulate the biosynthesis and secretion of this hormone.

Structure, function and expression of voltage-dependent sodium channels.

Voltage-dependent sodium channels control the transient inward current responsible for the action potential in most excitable cells. Members of this multigene family have been cloned, sequenced, and functionally expressed from various tissues and species, and common features of their structure have clearly emerged. Site-directed mutagenesis coupled with in vitro expression has provided additional insight into the relationship between structure and function. Subtle differences between sodium channel isoforms are also important, and aspects of the regulation of sodium channel gene expression and the modulation of channel function are becoming topics of increasing importance. Finally, sodium channel mutations have been directly linked to human disease, yielding insight into both disease pathophysiology and normal channel function. After a brief discussion of previous work, this review will focus on recent advances in each of these areas.

Chimeric study of sodium channels from rat skeletal and cardiac muscle.

Two isoforms of voltage-dependent Na channels, cloned from rat skeletal muscle, were expressed in Xenopus oocytes. The currents of rSkM1 and rSkM2 differ functionally in 4 properties: (i) tetrodotoxin (TTX) sensitivity, (ii) mu-conotoxin (mu-CTX) sensitivity, (iii) amplitude of single channel currents, and (iv) rate of inactivation. rSkM1 is sensitive to both TTX and mu-CTX. rSkM2 is resistant to both toxins. Currents of rSkM1 have a higher single channel conductance and a slower rate of inactivation than those of rSkM2. We constructed (i) chimeras by interchanging domain 1 (D1) between the two isoforms, (ii) block mutations of 22 amino acids in length that interchanged parts of the loop between transmembrane segments S5 and S6 in both D1 and D4, and (iii) point mutations in the SS2 region of this loop in D1. The TTX sensitivity could be switched between the two isoforms by the exchange of a single amino acid, tyrosine-401 in rSkM1 and cysteine-374 in rSkM2 in SS2 of D1. By contrast most chimeras and point mutants had an intermediate sensitivity to mu-CTX when compared with the wild-type channels. The point mutant rSkM1 (Y401C) had an intermediate single-channel conductance between those of the wild-type isoforms, whereas rSkM2 (C374Y) had a slightly lower conductance than rSkM2. The rate of inactivation was found to be determined by multiple regions of the protein, since chimeras in which D1 was swapped had intermediate rates of inactivation compared with the wild-type isoforms.

Molecular cloning and functional analysis of the promoter of rat skeletal muscle voltage-sensitive sodium channel subtype 2 (rSkM2): evidence for muscle-specific nuclear protein binding to the core promoter.

rSkM2 is a tetrodotoxin-resistant rat skeletal muscle voltage-sensitive sodium channel that is expressed in immature and denervated skeletal muscle and in adult heart. We have isolated a 3.7-kb gene segment that contains the first exon, multiple transcription initiation sites, the core promoter (nt -102 to +1), GC-rich elements (Sp1 recognition sites), three overlapping C-rich motifs (important for muscle-specific expression of some muscle genes), and multiple CANNTG (E-box) motifs (MyoD binding sites). A deletion analysis of the 5' upstream 2.8-kb segment, driving the rSkM2 core promoter, has localized a muscle-restrictive enhancer element (MRSE) at least 2 kb upstream from the core promoter. The core promoter is silenced by an additional cis element (-645/-506). The positive and negative cis-elements together drive transcription of the chloramphenicol acetyltransferase (CAT) reporter gene from the core promoter at about the same level as does the core promoter alone in a skeletal muscle differentiation stage-specific manner. Gel-shift assays have identified sequence- and cell-type-specific proteins that bind to a 16-bp region (-44/-29) containing C-rich motifs. Muscle-specific complexes formed from muscle cell nuclear extracts and a 16-bp element (-44/-29) are competed by unlabeled -44/-29 oligonucleotide but not by several mutant oligonucleotides that implicate nucleotides -40 to -38 and -34 to -32 in the binding of a nuclear protein (designated SkM2 transcription factor 1, SkM2-TF1). We conclude that rSkM2 gene expression depends on the interactions of positive and negative transcriptional regulators with tissue- and developmental stage-specific core promoter elements.

Effects of tyrosine-->phenylalanine mutations on auto- and trans-phosphorylation reactions catalyzed by the insulin receptor beta-subunit cytoplasmic domain.

Activation of the insulin receptor kinase is closely associated with autophosphorylation of several tyrosine residues in the cytoplasmic domain of the receptor's two beta-subunits. To determine the contribution of these tyrosine phosphorylations to autoactivation of the receptor kinase, we have blocked phosphorylation at specific tyrosine by replacing these tyrosine residues, individually and in combination, with phenylalanine in a soluble 45-kD analog of the cytoplasmic insulin receptor kinase domain (CIRK). Kinetic studies of auto- and transphosphorylation with this panel of mutated CIRKs indicate that: (i) None of the tyrosines (953, 960, 1,146, 1,150, 1,151, 1,316, or 1,322) are necessary for catalysis: all single Y-->F mutants retain the ability to autoactivate comparable to the parent CIRK. (ii) Two of the tyrosine autophosphorylation sites, either tyrosine 1,150 or 1,151, contribute most (70-80%) of the autoactivation, because replacement of these two tyrosines by phenylalanine was the minimal change that abolishes autoactivation. (iii) A mutant CIRK having all seven reported tyrosine phosphorylation sites replaced by phenylalanine retained basal kinase activity but was incapable of autoactivation. These findings imply that autoactivation can occur without phosphorylation having occurred at any single site (953, 960, 1,146, 1,150, 1,151, 1,316, or 1,322), and autophosphorylation need not follow an ordered, sequential pathway beginning, for example, at tyrosine 1,146 as proposed for the intact insulin receptor.

Pharmacological modulation of human cardiac Na+ channels.

Pharmacological modulation of human sodium current was examined in Xenopus oocytes expressing human heart Na+ channels. Na+ currents activated near -50 mV with maximum current amplitudes observed at -20 mV. Steady-state inactivation was characterized by a V1/2 value of -57 +/- 0.5 mV and a slope factor (k) of 7.3 +/- 0.3 mV. Sodium currents were blocked by tetrodotoxin with an IC50 value of 1.8 microM. These properties are consistent with those of Na+ channels expressed in mammalian myocardial cells. We have investigated the effects of several pharmacological agents which, with the exception of lidocaine, have not been characterized against cRNA-derived Na+ channels expressed in Xenopus oocytes. Lidocaine, quinidine and flecainide blocked resting Na+ channels with IC50 values of 521 microM, 198 microM, and 41 microM, respectively. Use-dependent block was also observed for all three agents, but concentrations necessary to induce block were higher than expected for quinidine and flecainide. This may reflect differences arising due to expression in the Xenopus oocyte system or could be a true difference in the interaction between human cardiac Na+ channels and these drugs compared to other mammalian Na+ channels. Importantly, however, this result would not have been predicted based upon previous studies of mammalian cardiac Na+ channels. The effects of DPI 201-106, RWJ 24517, and BDF 9148 were also tested and all three agents slowed and/or removed Na+ current inactivation, reduced peak current amplitudes, and induced use-dependent block. These data suggest that the alpha-subunit is the site of interaction between cardiac Na+ channels and Class I antiarrhythmic drugs as well as inactivation modifiers such as DPI 201-106.

Dual tandem promoter elements containing CCAC-like motifs from the tetrodotoxin-resistant voltage-sensitive Na+ channel (rSkM2) gene can independently drive muscle-specific transcription in L6 cells.

cis-Elements in the -129/+124 promoter segment of the rat tetrodotoxin-resistant voltage-gated sodium channel (rSkM2) gene that are responsible for reporter gene expression in cultured muscle cells were identified by deletion and scanning mutations. Nested 5' deletion constructs, assayed in L6 myotubes and NIH3T3 cells, revealed that the minimum promoter allowing muscle-specific expression is contained within the -57 to +1 segment relative to the major transcription initiation site. In the context of the -129/+1 construct, however, scanning mutations in the -69/+1 segment failed to identify any critical promoter elements. In contrast, identical mutations in a minimal promoter (-57/+124) showed that all regions except -29/-20 are essential for expression, especially the -57/-40 segment, consistent with the 5' deletion analysis. Further experiments showed that the distal (-129/-58) and proximal promoter (-57/+1) elements can independently drive reporter expression in L6 myotubes, but not in NIH3T3 fibroblasts. This pair of elements is similar in sequence and contains Sp1 sites (CCGCCC), CCAC-like motifs, but no E-boxes or MEF-2 sites. The two segments form similarly migrating complexes with L6 myotube nuclear extracts in gel-shift assays. Critical elements within the distal promoter element were defined by 10 base pair scanning mutations in the -119 to -60 region in the context of the -129/+1 segment containing a mutated -59/-50 segment that inactivates the proximal promoter. Nucleotides in the -119/-90 region, especially -109/-100, were the most important regions for distal promoter function. We conclude that the -129/+1 segment contains two tandem promoter elements, each of which can independently drive muscle-specific transcription. Supershifts with antibodies to Sp1 and myocyte nuclear factor (MNF) implicate the involvement of Sp1, MNF, and other novel factors in the transcriptional regulation of rSkM2 gene expression.

Enzymes of the beta-ketoadipate pathway in Pseudomonas putida: primary and secondary kinetic and equilibrium deuterium isotope effects upon the interconversion of (+)-muconolactone to cis,cis-muconate catalyzed by cis,cis-muconate cycloisomerase.

Primary and secondary kinetic and secondary equilibrium deuterium isotope effect studies on the cis,cis-muconate cycloisomerase catalyzed interconversion of cis,-cis-muconate (CCM) and (+)-muconolactone (ML) have been performed. The primary and solvent kinetic deuterium isotope effects upon Vmax for the reactions of (+)-[5R-2H]muconolactone in water (HOH) and (+)-muconolactone in deuterium oxide (DOD) to form cis,cis-muconate are about 2.5-2.6 with the heavier isotopic reactions being the slower ones. The secondary equilibrium isotope effect for the formation cis,-cis-[2,3,4,5-2H4] muconate from (+)-[2,3,4,5S-2H4]muconolactone is 1.32 for KH/KD = [( cis,cis-muconate]/[(+)-muconolactone])/ [(+)-[2,3,4,5S-2H4]muconolactone]) and agrees well with the measured value of 1.45 on the basis of the fumarase reaction [Cook, P. F., Blanchard, J. S., & Cleland, W. W. (1980) Biochemistry 19, 4853-4858]. The secondary kinetic deuterium isotope effect determined by the equilibrium perturbation method [Cleland, W. W. (1977) in Isotope Effects on Enzyme-Catalyzed Reactions (Cleland, W. W., O'Leary, M. H., & Northrop, D. B., Eds.) pp 153-175, University Park Press, Baltimore, MD] for the conversion of cis,cis-[2,3,4,5-2H4] muconate to (+)-[2,3,4,5S-2H4]muconolactone is 0.66, expressed as (VmaxCCM(H)/KmCCM(H]/(VmaxCCM(D)/KmCCM(D]. From the equilibrium and kinetic secondary deuterium isotope effects, the calculated value for the kinetic secondary deuterium isotope effect for the reverse reaction, (VmaxML(H)/KmML(H]/(VmaxML(D)/KmML(D], is about 0.96.(ABSTRACT TRUNCATED AT 250 WORDS)