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The tryptic peptides of rabbit muscle triose phosphate isomerase.

ABSTRACT: 1. The peptides obtained by tryptic digestion of S-[(14)C]carboxymethylated rabbit muscle triose phosphate isomerase have been studied. 2. The first step in the fractionation of the tryptic digest was gel filtration on coupled columns of Sephadex G-25 and G-50. Further fractionation was carried out by paper electrophoresis and paper chromatography. 3. The digest contained 26 peptides and three free amino acids. The sizes of the peptides ranged from two to 29 residues. 4. The sequences of the peptides have been determined. 5. The length of the polypeptide chains is about 250 amino acid residues. 6. The variant sequences encountered were due to partial deamidation; this may be one of the reasons for multiple forms of the enzyme. 7. The chicken and rabbit enzymes are compared. 8. Detailed evidence for the sequences of the tryptic peptides has been deposited as Supplementary Publication SUP 50024 at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1973) 131, 5.

SUBMITTER: Corran PH

PROVIDER: S-EPMC1166244 | biostudies-other | 1974 Apr

REPOSITORIES: biostudies-other

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The amino acid sequence of rabbit muscle triose phosphate isomerase.

Project description:The amino acid sequence of rabbit muscle triose phosphate isomerase was deduced by characterizing peptides that overlap the tryptic peptides. Thiol groups were modified by oxidation, carboxymethylation or aminoen. About 50 peptides that provided information about overlaps were isolated; the peptides were mostly characterized by their compositions and N-terminal residues. The peptide chains contain 248 amino acid residues, and no evidence for dissimilarity of the two subunits that comprise the native enzyme was found. The sequence of the rabbit muscle enzyme may be compared with that of the coelacanth enzyme (Kolb et al., 1974): 84% of the residues are in identical positions. Similarly, comparison of the sequence with that inferred for the chicken enzyme (Furth et al., 1974) shows that 87% of the residues are in identical positions. Limited though these comparisons are, they suggest that triose phosphate isomerase has one of the lowest rates of evolutionary change. An extended version of the present paper has been deposited as Supplementary Publication SUP 50040 (42 pages) at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1975) 145, 5.

| S-EPMC1165222 | biostudies-other

Refolding of triose phosphate isomerase.

Project description:The refolding and reactivation of the glycolytic enzyme triose phosphate isomerase (EC 5.3.1.1) has been studied. The enzyme, which is a dimer, is disaggregated and unfolded in solutions of guanidinium chloride. Unfolding, followed by changes in E(233), took place quite rapidly in 3m-guanidinium chloride (i.e. with a half-life of about 1 min). Refolding also took place rapidly when the solution was diluted about tenfold; two first-order processes could be resolved. Regain of enzymic activity was followed by diluting the solution of the denatured enzyme in guanidinium chloride into assay mixture. The half-life (i.e. the time when the activity was half the final activity) depended markedly on the concentration of protein at low concentrations (about 100ng/ml), but at higher concentrations the half-life became independent of concentration. Thus at low concentrations dimerization was a rate-determining step and this is taken to indicate that the monomers showed little or no activity under these conditions. The rate of regain of enzymic activity was the same as the rate of the slower process of refolding, which was detected spectroscopically. The native enzyme was resistant to proteolysis; high concentrations of subtilisin prevented regain of activity, but at lower concentrations refolding competed with proteolysis.

| S-EPMC1165801 | biostudies-literature

Amino acid sequences around the cysteine residues of rabbit muscle triose phosphate isomerase.

Project description:1. The nature of the subunits in rabbit muscle triose phosphate isomerase has been investigated. 2. Amino acid analyses show that there are five cysteine residues and two methionine residues/subunit. 3. The amino acid sequences around the cysteine residues have been determined; these account for about 75 residues. 4. Cleavage at the methionine residues with cyanogen bromide gave three fragments. 5. These results show that the subunits correspond to polypeptide chains, containing about 230 amino acid residues. The chains in triose phosphate isomerase seem to be shorter than those of other glycolytic enzymes.

| S-EPMC1176764 | biostudies-other

The active centre of rabbit muscle triose phosphate isomerase. The site that is labelled by glycidol phosphate.

Project description:1. Glycidol (2,3-epoxypropanol) phosphate is a specific irreversible inhibitor of rabbit muscle triose phosphate isomerase (EC 5.3.1.1); the site of attachment has now been studied. 2. The labelled enzyme was digested with pepsin and a modified peptide isolated. The sequence of the peptide is: Ala-Tyr-Glu-Pro-Val-Trp. 3. It is the glutamic acid residue in this peptide that is labelled: the peptide is thus a gamma-glutamyl ester derived from glycerol phosphoric acid. The same site is labelled by a mixture of glycidol and inorganic phosphate. 4. Kinetic and stereochemical features of these reactions are discussed.

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Specificity and kinetics of triose phosphate isomerase from chicken muscle.

Project description:The isolation of crystalline triose phosphate isomerase from chicken breast muscle is described. The values of k(cat.) and K(m) for the reaction in each direction were determined from experiments over wide substrate-concentration ranges, and the reactions were shown to obey simple Michaelis-Menten kinetics. With d-glyceraldehyde 3-phosphate as substrate, k(cat.) is 2.56x10(5)min(-1) and K(m) is 0.47mm; with dihydroxyacetone phosphate as substrate, k(cat.) is 2.59x10(4)min(-1) and K(m) is 0.97mm. The enzyme-catalysed exchange of the methyl hydrogen atoms of the ;virtual substrate' monohydroxyacetone phosphate with solvent (2)H(2)O or (3)H(2)O was shown. This exchange is about 10(4)-fold slower than the corresponding exchange of the C-3 hydrogen of dihydroxyacetone phosphate. The other deoxy substrate, 3-hydroxypropionaldehyde phosphate, was synthesized, but is too unstable in aqueous solution for analogous proton-exchange reactions to be studied.

| S-EPMC1174080 | biostudies-other

Characterization of stress and methylglyoxal inducible triose phosphate isomerase (OscTPI) from rice.

Project description:As compared with plant system, triose phosphate isomerase (TPI), a crucial enzyme of glycolysis, has been well studied in animals. In order to characterize TPI in plants, a full-length cDNA encoding OscTPI was cloned from rice and expressed in E. coli. The recombinant OscTPI was purified to homogeneity and it showed Km value of 0.1281 ± 0.025 µM, and the Vmax value of 138.7 ± 16 µmol min (-1) mg (-1) which is comparable to the kinetic values studied in other plants. The OscTPI was found to be exclusively present in the cytoplasm when checked with the various methods. Functional assay showed that OscTPI could complement a TPI mutation in yeast. Real time PCR analysis revealed that OscTPI transcript level was regulated in response to various abiotic stresses. Interestingly, it was highly induced under different concentration of methylglyoxal (MG) stress in a concentration dependent manner. There was also a corresponding increase in the protein and the enzyme activity of OscTPI both in shoot and root tissues under MG stress. Our result shows that increases in MG leads to the increase in TPI which results in decrease of DHAP and consequently decrease in the level of toxic MG.

| S-EPMC3493422 | biostudies-literature

Leishmania donovani triose phosphate isomerase: a potential vaccine target against visceral leishmaniasis.

Project description:Visceral leishmaniasis (VL) is one of the most important parasitic diseases with approximately 350 million people at risk. Due to the non availability of an ideal drug, development of a safe, effective, and affordable vaccine could be a solution for control and prevention of this disease. In this study, a potential Th1 stimulatory protein- Triose phosphate isomerase (TPI), a glycolytic enzyme, identified through proteomics from a fraction of Leishmania donovani soluble antigen ranging from 89.9-97.1 kDa, was assessed for its potential as a suitable vaccine candidate. The protein- L. donovani TPI (LdTPI) was cloned, expressed and purified which exhibited the homology of 99% with L. infantum TPI. The rLdTPI was further evaluated for its immunogenicity by lymphoproliferative response (LTT), nitric oxide (NO) production and estimation of cytokines in cured Leishmania patients/hamster. It elicited strong LTT response in cured patients as well as NO production in cured hamsters and stimulated remarkable Th1-type cellular responses including IFN-ã and IL-12 with extremely lower level of IL-10 in Leishmania-infected cured/exposed patients PBMCs in vitro. Vaccination with LdTPI-DNA construct protected naive golden hamsters from virulent L. donovani challenge unambiguously (?90%). The vaccinated hamsters demonstrated a surge in IFN-ã, TNF-á and IL-12 levels but extreme down-regulation of IL-10 and IL-4 along with profound delayed type hypersensitivity and increased levels of Leishmania-specific IgG2 antibody. Thus, the results are suggestive of the protein having the potential of a strong candidate vaccine.

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The active centre of triose phosphate isomerase.

Project description:The molecular weight and amino acid composition of triose phosphate isomerase have been determined. The molecular weight (43000) is lower and the molecular activity (500000) higher than those of most other glycolytic enzymes. Reaction with iodoacetate (studied with radioactive reagent) takes place in two phases: in the first phase, at pH6.3, cysteine and methionine groups react and enzymic activity is unimpaired; in the second phase, histidine reacts and enzymic activity is lost. Photo-oxidation leads to inactivation, with loss of cysteine, of histidine and of tryptophan, but little loss of tyrosine. The mechanism postulated for the action of the enzyme demands the intervention of a group functioning as a base, and the results obtained are consistent with histidine's being the basic group in the active centre.

| S-EPMC1265204 | biostudies-other

Studies of triose phosphate isomerase by hydrogen exchange.

Project description:The (3)H-H exchange of chicken muscle and rabbit muscle triose phosphate isomerases was studied. Their behaviour was mostly very similar. ;Exchange-in' (acquisition of radioactivity when protein was incubated in (3)H(2)O) was measured at 37 degrees C and at pH7.5, and the rates of exchange of the native and liganded enzymes were compared. Inhibitors and substrates retarded exchange, substrates showing the most marked effect; structural rearrangements in the enzyme may thus play some part in catalysis. The inhibitor phosphoglycollate affected the rabbit enzyme, but had little or no effect on the chicken enzyme. ;Exchange-out' (loss of radioactivity from protein previously labelled by incubation in (3)H(2)O) was measured by hollow-fibre dialysis. When ligand was removed during the course of dialysis (by replacing buffer that contained ligand with buffer that lacked ligand) there was a prompt decrease in the number of labelled H atoms of the protein. Analysis of the curves provides some information about the number and half-lives of the responsive H atoms. Ligands decrease the motility of the protein and affect about one-fifth of the chain. Low concentrations of glycerol 3-phosphate have an effect that is greater than expected.

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pH-dependence of the triose phosphate isomerase reaction.

Project description:The pH-dependences of the kinetic parameters k(cat.) and K(m) for the triose phosphate isomerase reaction were determined in each direction. Apparent pK(a) values of 6.0 and 9.0 are observed in the dependences of k(cat.)/K(m). The pH-dependences of k(cat.) are sigmoid, with apparent pK(a) values of about 6.0. The results are interpreted in terms of a single base on the enzyme providing an efficient proton-shuttling mechanism for the isomerization.

| S-EPMC1174081 | biostudies-other

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