Calmodulin-dependent protein kinase II from bovine cardiac muscle: purification and differential activation by calcium

Calmodulin-dependent protein kinase II was purified to apparent homogeneity with a high yield from the total calmodulin-binding protein fraction of bovine cardiac muscle in a single step by gel filtration column chromatography. This procedure is simple and suitable for adaptation to large scale preparations. The purified calmodulin-dependent protein kinase has a specific enzymic activity of 2.4 mumol/min/mg when mixed histone was used as a substrate. The preparation of enzyme appears to be homogeneous when examined by SDS-PAGE. The molecular weight of the enzyme was determined to be 570 kDa by gel filtration. SDS-PAGE of the enzyme subunit showed a single protein band with an apparent molecular weight of 56 kDa. These results suggest that the calmodulin-dependent protein kinase II from bovine heart is composed of 10 identical subunits. Anti-peptide antibody raised against multifunctional calmodulin-dependent protein kinase II from rat brain showed a single immunoreactive band of 56 kDa on Western blot. These results suggested that bovine cardiac muscle calmodulin-dependent protein kinase could resemble the brain isozyme. Calmodulin-dependent protein kinase II undergoes autophosphorylation with a maximal incorporation of 1 mol of phosphate per mol of the subunit of the enzyme and the autophosphorylated enzyme remains active in the absence of Ca2+ and calmodulin. The concentration of Ca2+ required for the activation of calmodulin-dependent protein kinase II depends on the level of calmodulin in the reaction.     

Baculovirus-mediated expression and characterization of the full-length murine DNA methyltransferase

The original cDNA sequence reported for the murine DNA methyltransferase (MTase) was not full length. Recently, additional cDNA sequences have been reported that lie upstream of the original and contain an extended open reading frame with three additional ATGs in frame with the coding region [Tucker et al . (1996) Proc. Natl. Acad. Sci. USA , 93, 12920-12925; Yoder et al . (1996) J. Biol. Chem . 271, 31092-31097]. Genomic DNA upstream of this ATG contains two more ATGs in frame and no obvious splice site. We have constructed, and expressed in baculovirus, MTase clones that begin at each of these four ATGs and examined their properties. Constructs beginning with any of the first three ATGs as their initiator methionines give a predominant DNA MTase band of approximately 185 kDa on SDS-PAGE corresponding to translational initiation at the third ATG. The fourth ATG construct gives a much smaller protein band of 173 kDa. The 185 kDa protein was purified by HPLC, characterized by mass spectrometry and has a measured molecular mass of 184 +/- 0.5 kDa. All of these MTases were functional in vitro and steady state kinetic analysis showed that the recombinant proteins exhibit similar kinetic properties irrespective of their length. The homogeneous recombinant enzyme from the fourth ATG construct shows a 2.5-fold preference for a hemi-methylated DNA substrate as compared to an unmethylated substrate, whereas the 185 kDa protein is equally active on both substrates. The kinetic properties of the recombinant enzyme are similar to those reported for the native MTase derived from murine erythroleukemia cells. The new clones are capable of yielding large quantities of intact MTases for further structural and functional studies.     

Cloning, sequencing, and expression of the gene encoding cyclic 2, 3-diphosphoglycerate synthetase, the key enzyme of cyclic 2, 3-diphosphoglycerate metabolism in Methanothermus fervidus

Cyclic 2,3-diphosphoglycerate synthetase (cDPGS) catalyzes the synthesis of cyclic 2,3-diphosphoglycerate (cDPG) by formation of an intramolecular phosphoanhydride bond in 2,3-diphosphoglycerate. cDPG is known to be accumulated to high intracellular concentrations (>300 mM) as a putative thermoadapter in some hyperthermophilic methanogens. For the first time, we have purified active cDPGS from a methanogen, the hyperthermophilic archaeon Methanothermus fervidus, sequenced the coding gene, and expressed it in Escherichia coli. cDPGS purification resulted in enzyme preparations containing two isoforms differing in their electrophoretic mobility under denaturing conditions. Since both polypeptides showed the same N-terminal amino acid sequence and Southern analyses indicate the presence of only one gene coding for cDPGS in M. fervidus, the two polypeptides originate from the same gene but differ by a not yet identified modification. The native cDPGS represents a dimer with an apparent molecular mass of 112 kDa and catalyzes the reversible formation of the intramolecular phosphoanhydride bond at the expense of ATP. The enzyme shows a clear preference for the synthetic reaction: the substrate affinity and the Vmax of the synthetic reaction are a factor of 8 to 10 higher than the corresponding values for the reverse reaction. Comparison with the kinetic properties of the electrophoretically homogeneous, apparently unmodified recombinant enzyme from E. coli revealed a twofold-higher Vmax of the enzyme from M. fervidus in the synthesizing direction.     

Expression of neurotrophin-3 in the growing velvet antler of the red deer Cervus elaphus

Adenine phosphoribosyltransferase (APRT) from Saccharomyces cerevisiae was purified approximately 1500-fold. The enzyme catalyzes the Mg-dependent condensation of adenine and 5-phosphoribosylpyrophosphate (PRPP) to yield AMP. The purification procedure included anion exchange chromatography, chromatofocusing and gel filtration. Elution of the enzyme from the chromatofocusing column indicated a pI value of 4.7. The molecular mass for the native enzyme was 50 kDa; however, upon electrophoresis under denaturing conditions two bands of apparent molecular mass of 29 and 20 kDa were observed. We have previously reported the presence of two separate coding sequences for APRT, APT1 and APT2 in S. cerevisiae. The appearance of two bands under denaturing conditions suggests that, unlike other APRTs, this enzyme could form heterodimers. This may be the basis for substrate specificity differences between this enzyme and other APRTs. Substrate kinetics and product inhibition patterns are consistent with a ping-pong mechanism. The Km for adenine and PRPP were 6 microM and 15 microM, respectively and the Vmax was 15 micromol/min. These kinetic constants are comparable to the constants of APRT from other organisms.     

Purification and properties of human D-3-hydroxyacyl-CoA dehydratase: medium-chain enoyl-CoA hydratase is D-3-hydroxyacyl-CoA dehydratase

Human medium-chain enoyl-CoA hydratase was purified from liver, because we noticed the presence of a high medium-chain enoyl-CoA hydratase activity in human skin fibroblasts catalyzed by an enzyme different from the known enzymes catalyzing the enoyl-CoA hydratase reaction. Two enzyme preparations were obtained. One of them, preparation I, consisted of 46-kDa polypeptide, and its molecular mass was estimated to be 86 kDa. The other, preparation II, consisted of a major 77-kDa polypeptide and minor smaller polypeptides including 46-kDa polypeptide. The molecular mass of preparation II was 154 kDa. Both enzyme preparations catalyzed reversible dehydration of medium-chain D-3-hydroxyacyl-CoA to 2-trans-enoyl-CoA, but did not react with L-3-hydroxyacyl-CoA. Catalytic properties and immunochemical reactivities of these enzyme preparations were nearly the same. The cross-reactive material to the antibody was confirmed to be in peroxisomes by immunohistochemical study of cultured human skin fibroblasts.     

Immunoassays fail to detect antibodies against neuronal calcium channels in amyotrophic lateral sclerosis serum

A NAD-dependent enzyme that catalyzes the oxidation of retinal to retinoic acid has been purified to homogeneity from bovine kidney. The procedures used in the purification included ion-exchange chromatography on DEAE-Sepharose, affinity chromatography on Affi-gel blue and chromatography on a Mono-Q anion-exchange column. On the Mono-Q column, the enzyme aldehyde dehydrogenase (ALDH) resolved into two activity peaks designated as ALDH1 and ALDH2. The enzymes ALDH1 and ALDH2 were purified about 114- and 65-fold, respectively. Gel filtration chromatography of the partially purified native enzyme on Sephacryl S-200 HR exhibited a molecular mass of about 108 kDa. Electrophoresis of the purified enzymes under nondenaturing conditions showed a single protein band. However, sodium dodecyl sulfate--polyacrylamide gel electrophorsis indicated three protein bands in the 55, 30, and 22 kDa molecular mass regions. Both enzymes exhibited a broad substrate specificity oxidizing a wide variety of aliphatic and aromatic aldehydes. The ALDH1 enzyme had a pI of 7.45 and exhibited a low Km (6.37 microM) for retinal, while the ALDH2 enzyme was found to have very low Km for acetaldehyde (0.98 microM). Based on its kinetic properties, it is suggested that the ALDH1 enzyme may be the primary enzyme for oxidizing retinal to retinoic acid in bovine kidney.     

Iron-sulfur clusters/semiquinones in complex I

NADH-quinone 1 oxidoreductase (Complex I) isolated from bovine heart mitochondria was, until recently, the major source for the study of this most complicated energy transducing device in the mitochondrial respiratory chain. Complex I has been shown to contain 43 subunits and possesses a molecular mass of about 1 million. Recently, Complex I genes have been cloned and sequenced from several bacterial sources including Escherichia coli, Paracoccus denitrificans, Rhodobacter capsulatus and Thermus thermophilus HB-8. These enzymes are less complicated than the bovine enzyme, containing a core of 13 or 14 subunits homologous to the bovine heart Complex I. From this data, important clues concerning the subunit location of both the substrate binding site and intrinsic redox centers have been gleaned. Powerful molecular genetic approaches used in these bacterial systems can identify structure/function relationships concerning the redox components of Complex I. Site-directed mutants at the level of bacterial chromosomes and over-expression and purification of single subunits have allowed detailed analysis of the amino acid residues involved in ligand binding to several iron-sulfur clusters. Therefore, it has become possible to examine which subunits contain individual iron-sulfur clusters, their location within the enzyme and what their ligand residues are. The discovery of g=2.00 EPR signals arising from two distinct species of semiquinone (SQ) in the activated bovine heart submitochondrial particles (SMP) is another line of recent progress. The intensity of semiquinone signals is sensitive to DeltamicroH+ and is diminished by specific inhibitors of Complex I. To date, semiquinones similar to those reported for the bovine heart mitochondrial Complex I have not yet been discovered in the bacterial systems. This mini-review describes three aspects of the recent progress in the study of the redox components of Complex I: (A) the location of the substrate (NADH) binding site, flavin, and most of the iron-sulfur clusters, which have been identified in the hydrophilic electron entry domain of Complex I; (B) experimental evidence indicating that the cluster N2 is located in the amphipathic domain of Complex I, connecting the promontory and membrane parts. Very recent data is also presented suggesting that the cluster N2 may have a unique ligand structure with an atypical cluster-ligation sequence motif located in the NuoB (NQO6/PSST) subunit rather than in the long advocated NuoI (NQO9/TYKY) subunit. The latter subunit contains the most primordial sequence motif for two tetranuclear clusters; (C) the discovery of spin-spin interactions between cluster N2 and two distinct Complex I-associated species of semiquinone. Based on the splitting of the g1 signal of the cluster N2 and concomitant strong enhancement of the semiquinone spin relaxation, one semiquinone species was localized 8-11 A from the cluster N2 within the inner membrane on the matrix side (N-side). Spin relaxation of the other semiquinone species is much less enhanced, and thus it was proposed to have a longer distance from the cluster N2, perhaps located closer to the other side (P-side) surface of the membrane. A brief introduction of EPR technique was also described in Appendix A of this mini-review.     

Pre-steady state kinetic analysis of an enzymatic reaction monitored by time-resolved electrospray ionization mass spectrometry

For the first time, the new technique of time-resolved electrospray ionization mass spectrometry (ESI-MS) has been used to accurately measure the pre-steady state kinetics of an enzymatic reaction by monitoring a transient enzyme intermediate. The enzyme used to illustrate this approach, Bacillus circulans xylanase, is a retaining glycosidase that hydrolyzes xylan or beta-xylobiosides through a double-displacement mechanism involving a covalent xylobiosyl-enzyme intermediate. A low steady state level of this intermediate formed during the hydrolysis of 2,5-dinitrophenyl beta-d-xylobioside was detected by time-resolved ESI-MS. The low concentration of this intermediate and its rate of formation did not permit pre-steady state kinetic analysis. By contrast, the covalent intermediate accumulates fully when the Tyr80Phe mutant hydrolyzes the same substrate. Using time-resolved ESI-MS, the pre-steady state kinetic parameters for the formation of the covalent intermediate in the mutant xylanase have been determined. The kinetic data are in agreement with those determined by monitoring the release of 2, 5-dinitrophenol with stopped-flow UV-vis spectroscopy. This demonstrates that time-resolved ESI-MS can be used to accurately monitor the pre-steady state kinetics of enzymatic reactions, with the advantage of identifying transient enzyme intermediates by their mass.     

Kinetics of slow reversible inhibition of human muscle creatine kinase by planar anions

The toxicity of NO3- and NO2- to mammals has been widely publicized. However, the kinetic mechanism of inhibition of human muscle creatine kinase by NO3- and NO2- has not been explored. The kinetic theory of the substrate reaction during the modification of enzyme activity previously described by Tsou (Adv. Enzymol. Related Areas Mol. Biol. 1988, 61, 381-436) has been applied to a study of the kinetics of slow reversible inhibition of human muscle creatine kinase by planar anions (NO3- and NO2-). The kinetic equation of the substrate reaction was derived from theoretical analysis and experimental data, then simplified. The microscopic rate constants for the reaction of the inhibitors with the enzyme were obtained from the simplified equation for the substrate reaction in the presence of the inhibitors. The results show that the apparent forward rate constant A is dependent on ATP concentration, indicating competition between the inhibitor (NO3- or NO2-) and ATP. The results also suggest that binding of creatine-MgADP and the anion with the enzyme is very tight, since their binding constants are much higher than those for normal substrates.     

Reversal of cyanide inhibition of cytochrome c oxidase by the auxiliary substrate nitric oxide: an endogenous antidote to cyanide poisoning?

Nitric oxide (NO) is shown to overcome the cyanide inhibition of cytochrome c oxidase in the presence of excess ferrocytochrome c and oxygen. Addition of NO to the partially reduced cyanide-inhibited form of the bovine enzyme is shown by electron paramagnetic resonance spectroscopy to result in substitution of cyanide at ferriheme a3 by NO with reduction of the heme. The resulting nitrosylferroheme a3 is a 5-coordinate structure, the proximal bond to histidine having been broken. NO does not simply act as a reversibly bound competitive inhibitor but is an auxiliary substrate consumed in a catalytic cycle along with ferrocytochrome c and oxygen. The implications of this observation with regard to estimates of steady-state NO levels in vivo is discussed. Given the multiple sources of NO available to mitochondria, the present results appear to explain in part some of the curious biomedical observations reported by other laboratories; for example, the kidneys of cyanide poisoning victims surprisingly exhibit no significant irreversible damage, and lethal doses of potassium cyanide are able to inhibit cytochrome c oxidase activity by only approximately 50% in brain mitochondria.     

Versican V2 is a major extracellular matrix component of the mature bovine brain

We have isolated and characterized the proteoglycan isoforms of versican from bovine brain extracts. Our approach included (i) cDNA cloning and sequencing of the entire open reading frame encoding the bovine versican splice variants; (ii) preparation of antibodies against bovine versican using recombinant core protein fragments and synthetic peptides; (iii) isolation of versican isoforms by ammonium sulfate precipitation followed by anion exchange and hyaluronan affinity chromatography; and (iv) characterization by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining or immunoblotting. Our results demonstrate that versican V2 is, together with brevican, a major component of the mature brain extracellular matrix. Versicans V0 and V1 are only present in relatively small amounts. Versican V2 migrates after chondroitinase ABC digestion with an apparent molecular mass of about 400 kDa, whereas it barely enters a 4-15% polyacrylamide gel without the enzyme treatment. The 400-kDa product is recognized by antibodies against the glycosaminoglycan-alpha domain and against synthetic NH2- and COOH-terminal peptides. Our preparations contain no major proteolytic products of versican, e.g. hyaluronectin or glial hyaluronate-binding protein. Having biochemical quantities of versican V2 available will allow us to test its putative modulatory role in neuronal cell adhesion and axonal growth.     

Molecular and phylogenetic characterization of isopropylmalate dehydrogenase of a thermoacidophilic archaeon, Sulfolobus sp. strain 7

The archaeal leuB gene encoding isopropylmalate dehydrogenase of Sulfolobus sp. strain 7 was cloned, sequenced, and expressed in Escherichia coli. The recombinant Sulfolobus sp. enzyme was extremely stable to heat. The substrate and coenzyme specificities of the archaeal enzyme resembled those of the bacterial counterparts. Sedimentation equilibrium analysis supported an earlier proposal that the archaeal enzyme is homotetrameric, although the corresponding enzymes studied so far have been reported to be dimeric. Phylogenetic analyses suggested that the archaeal enzyme is homologous to mitochondrial NAD-dependent isocitrate dehydrogenases (which are tetrameric or octameric) as well as to isopropylmalate dehydrogenases from other sources. These results suggested that the present enzyme is the most primitive among isopropylmalate dehydrogenases belonging in the decarboxylating dehydrogenase family.     

Porcine pyridoxal kinase c-DNA cloning, expression and confirmation of its primary sequence

Porcine brain pyridoxal kinase has been cloned. A 1.2 kilo-based cDNA with a 966-base pair open reading frame was determined from a porcine brain cortex cDNA library using PCR technique. The DNA sequence was shown to encode a protein of 322 amino acid residues with a molecular mass of 35.4 kDa. The amino acid sequence deduced from the nucleotide sequence of the cDNA was shown to match the partial primary sequence of pyridoxal kinase. Expression of the cloned cDNA in E. coli has produced a protein which displays both pyridoxal kinase activity and immunoreactivity with monoclonal antibodies raised against natural enzyme from porcine brain. With respect to the physical properties, it is shown that the recombinant protein exhibits identical kinetic parameters with the pure enzyme from porcine brain. Although the primary sequence of porcine pyridoxal kinase has been shown to share 87% homology with the human enzyme, we have shown that the porcine enzyme carries an extra peptide of ten amino acid residues at the N-terminal domain.     

Purification and characterization of ovine pancreatic elastase

Elastase was isolated from ovine pancreas and purified to homogeneity by two different procedures. One involved precipitation with ammonium sulphate, p-aminobenzamidine-Sepharose chromatography, CM-Sepharose ion exchange chromatography and S-300 Sephacryl chromatography. The other involved the direct adsorption of elastase by tri-L-alanyl-Sepharose chromatography and a CM-Sepharose step. The enzyme, which was produced in an inactive form in the pancreas, was activated with a trace of trypsin prior to chromatography. Ovine pancreatic elastase has an isoelectric point above pI 9.3 and its molecular mass is estimated at approximately 25 kDa. The optimal pH range for activity is between 8.0 and 8.4 and the enzyme is unstable at pH below 4.0 and above 10.0 and at temperatures above 65 degrees C. The kinetic properties of the enzyme were determined with succinyl-Ala-Ala-Ala-p-nitroanilide as the substrate. Km and kcat Km-1 proved to be similar to the kinetic parameters of porcine elastase determined simultaneously.     

Kinetics of inactivation of green crab (Scylla Serrata) alkaline phosphatase during removal of zinc ions by ethylenediaminetetraacetic acid disodium

Green crab (Scylla Serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, the each active site in which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou has been applied to a study on the kinetics of the course of inactivation of the enzyme by ethylenediaminetetraacetic acid disodium (EDTA). The kinetics of the substrate reaction with different concentrations of the substrate p-nitrophenyl phosphate (PNPP) and inactivator EDTA suggested a complexing mechanism for inactivation by, and substrate competition with, EDTA at the active site. The inactivation kinetics are single phasic, showing the initial formation of an enzyme-EDTA complex is a relatively rapid reaction, followed a slow inactivation step that probably involves a conformational change of the enzyme. Zinc ions are finally removed from the enzyme. The presence of metal ions apparently stabilizes an active-site conformation required for enzyme activity.     

Diastereomeric specificity of 2',3'-cyclic nucleotide 3'-phosphodiesterase

The diastereomers of adenosine and uridine 2',3'-cyclic phosphorothioates were tested as substrates for 2',3'-cyclic nucleotide 3'-phosphodiesterase from bovine brain. The enzyme cleaves the Sp (or exo) diastereomers efficiently, whereas the Rp (or endo) diastereomers are resistant to hydrolysis, even after long incubation. As the enzyme exhibits strong substrate inhibition the precise determination of kinetic parameters posed problems, particularly with phosphorothioates. The stereoselectivity of this enzyme is opposite to that of RNase T1 and RNase A and thus could be a useful complement in determination of the configuration of nucleoside 2',3'-cyclic phosphorothioates resulting from hydrolysis reactions of unknown stereochemical course.     

Kinetic competence of a phosphoryl enzyme intermediate in the glucose-1,6-p2 synthase-catalyzed reaction. Purification, properties, and kinetic studies

Glucose-1,6-P2 synthase of beef brain which catalyzes the formation of glucose-1,6-P2 and glycerate-3-P from glycerate-1,3-P2 and glucose-1-P has been purified 700-fold with an overall recovery of 19%. The purification procedure involves an ammonium sulfate fractionation of the crude extract, DE52 and hydroxylapatite column chromatography and isoelectric focusing. The isolated enzyme appears to be homogeneous by sodium dodecyl sulfate gel electrophoresis. Its molecular weight is estimated to be about 70,000 by gel filtration on Sephadex G-200 which agrees with the value obtained by sodium dodecyl sulfate gel electrophoresis. A phosphoryl enzyme intermediate in the catalytic reaction is indicated by the following evidence: glycerate-1,3-P2[1-32P] labels the enzyme. The label is removed by acceptor substrates such as glucose-1-P. Using a rapid quenching device at 23 degrees and pH 8.0, the first order rate constant for phosphorylation of the enzyme is 20 s-1, compared with an overall rate with the best acceptor, glucose-1-P, of 19 s-1. Dephosphorylation by glucose-1-P is at 37 s-1. Mg2+ is required for both phosphoryl transfers and the overall reaction. In the complete reaction the fraction of enzyme that is phosphorylated depends on the concentrations of glycerate-1,3-P2 and the concentration and nature of the acceptor in a way that could be predicted from the steady state parameters, the Km values, and the kinetic constants observed for the single turnover. Reciprocal plots of initial rates as a function of both substrate concentrations are families of parallel lines. The 32P-labeled phosphoryl enzyme intermediate was found to be acid-stable and somewhat alkaline-labile. Phosphoserine was identified from the partial acid hydrolysate of a protease digest of [32P] phosphoryl enzyme by two-dimensional thin layer chromatography.     

Purification, characterization, and mechanism of a flavin mononucleotide-dependent 2-nitropropane dioxygenase from Neurospora crassa

A nitroalkane-oxidizing enzyme was purified to homogeneity from Neurospora crassa. The enzyme is composed of two subunits; the molecular weight of each subunit is approximately 40,000. The enzyme catalyzes the oxidation of nitroalkanes to produce the corresponding carbonyl compounds. It acts on 2-nitropropane better than on nitroethane and 1-nitropropane, and anionic forms of nitroalkanes are much better substrates than are neutral forms. The enzyme does not act on aromatic compounds. When the enzyme reaction was conducted in an 18O2 atmosphere with the anionic form of 2-nitropropane as the substrate, acetone (with a molecular mass of 60 Da) was produced. This indicates that the oxygen atom of acetone was derived from molecular oxygen, not from water; hence, the enzyme is an oxygenase. The reaction stoichiometry was 2CH3CH(NO2)CH3 + O2-->2CH3COCH3 + 2HNO2, which is identical to that of the reaction of 2-nitropropane dioxygenase from Hansenula mrakii. The reaction of the Neurospora enzyme was inhibited by superoxide anion scavengers in the same manner as that of the Hansenula enzyme. Both of these enzymes are flavoenzymes; however, the Neurospora enzyme contains flavin mononucleotide as a prosthetic group, whereas the Hansenula enzyme contains flavin adenine dinucleotide.     

Effect of x-irradiation on properties of Ca2+-calmodulin-dependent protein kinase from rat spleen lymphocytes

Ca(2+)-calmodulin-dependent kinase has been isolated and purified from rat spleen lymphocytes cytosol in control and 12 h after the effect of X-ray radiation in a dose of 0.5 and 1 Gy. The isolated enzyme showed a remarkable similar substrate specificity and kinetic properties to those of rat brain and rat spleen calmodulin-dependent protein kinase II.     

Purification, crystallization and properties of triosephosphate isomerase from human skeletal muscle

1. Triosephosphate isomerase (D-glyceraldehyde-3-phosphate ketoisomerase, EC 5.3.1.1) from human skeletal muscle was purified to homogeneity and crystallized. The crystalline enzyme preparation was resolved on polyacrylamide-gel electrophoresis into three isoenzymes. 2. The molecular weight of the enzyme estimated by gel filtration method was found to be 57,400 +/- 3000. Molecular weight determination under dissociation conditions indicated a dimeric subunit structure of the enzyme. 3. The apparent Km for D-glyceraldehyde-3-phosphate as substrate is 0.34 mM, and for dihydroxyacetone phosphate, 0.61 mM. Vmax of the reaction is, respectively, 7200 and 660 units/mg protein at 25 degrees C and pH 7.5. 4. Molecular and kinetic properties of triosephosphate isomerase from human skeletal muscle are very similar to those of rabbit muscle enzyme.