Origin of the triosephosphate isomerase isozymes in humans: genetic evidence for the expression of a single structural locus.

A genetic approach is used to ascertain that a single structural locus for triosephosphate isomerase (TPI) (E.C.5.3.1.1.) is expressed in rapidly dividing human lymphoblasts. This approach is made possible through the identification of a rare electrophoretic variant of human TPI. The variant phenotype is expressed by the TPI-B isozyme in both erythrocytes and peripheral lymphocytes. The variant phenotype is also expressed in the thermostability and electrophoretic pattern of the TPI-A isozyme in mitogen-stimulated lymphoblasts, indicating that TPI-A and TPI-B are products of the same structural locus. These findings are in contrast to the recent conclusions of Yuan et al. based upon structural analysis, suggesting that the TPI-A and TPI-B isozymes are products of distinct structural loci.     

Cell proliferation-associated expression of a recently evolved isozyme of triosephosphate isomerase

An electrophoretically unique, thermolabile isozyme of triosephosphate isomerase (TPI; EC 5.3.1.1) accounts for 10–30% of the enzymatic activity in a range of mitotically active human cells and tissues. This type 2 form (subunit) of human TPI appears in two isozymes, an anodally migrating, relative to the constitutive TPI-1/1 homodimer, TPI-2/2 homodimer and the TPI-1/2 heterodimer with an intermediate mobility. Human cell types expressing the induced isozyme, which is the product of the same structural locus as the constitutive isozyme, include mitogen-stimulated lymphocytes, virally transformed B-lymphoblastoid cells, leukemia-derived T-lymphoblastoid cells, HeLa cells, both normal and transformed fibroblasts, and placental tissue. Extracts of nondividing or terminally differentiated human cells/tissues, such as erythrocytes, striated muscle, peripheral lymphocytes, and platelets, contain high levels of the constitutive TPI-1/1 isozyme but little or undetectable levels of the TPI-1/2 or TPI-2/2 isozyme. The cell division-associated TPI-1/2 and -2/2 isozymes are distinct in electrophoretic mobility from the deamidated forms of the constitutive isozyme. Extracts of dividing gorilla fibroblasts display an isozyme pattern identical to that of proliferating human cells, but various proliferating cells derived from the African green monkey, rabbit, and chicken express only the constitutive isozyme. Thus, expression of the cell division-associated isozyme of TPI is restricted to the hominoids, suggesting a recently evolved modification mechanism which is specifically activated in proliferating cells.Financial support was derived from Contract EY-77-C-02-2828 from the Department of Energy and Training Grant 5-T32-GM07544 from the National Institutes of Health.     

Inheritance and subcellular localization of triose-phosphate isomerase in dwarf mountain pine (Pinus mugo).

Several trees with expected heterozygous phenotype for triose-phosphate isomerase (TPI) were discovered in a population of dwarf mountain pine (Pinus mugo Turra) from southern Poland. As the inheritance of this enzyme in pines has not been reported, segregation of allelic variants was tested in eight trees with putative heterozygous phenotypes for two loci, TpiA and TPIB: Linkage between these and some other isozyme loci were studied and evidence for linkage has been found between TpiA and PgdA (r = 0.10) and between TpiB and DiaD (r = 0.36), but in single trees only. The subcellular localization of TPI isozymes was determined by comparing isoenzymes from the total extract with those found in fraction enriched in plastids, prepared by differential gradient centrifugation of cellular organelles. The more slowly migrating TPI-B isozyme is located in plastids.     

Hominoid triosephosphate isomerase: Characterization of the major cell proliferation specific isozyme

Summary Proliferating cells derived from hominoid species contain electrophoretically separable forms of triosephosphate isomerase (TPI), including a constitutive isozyme and major and minor cell proliferation specific isozymes. Genetic studies have shown that the constitutive and inducible isozymes are products of the same structural gene. A procedure has been developed for the rapid isolation of the constitutive and major proliferation specific TPI isozymes from human lymphoblastoid B cells. [35^S]methionine labeled isozymes were purified through several steps of polyacrylamide gel electrophoresis in sufficient quantities for turnover studies and preliminary structural analysis. The intact isozymes were subjected to 23 steps of automated Edman degradation; both preparations yield a [³⁵S] PTH-methionine only at cycle 14, as expected if the protein is TPI. Neither isozyme contains an blocked NH₂-terminus and length heterogenity at the amino terminal does not exist. A comparison of the two purified isozymes on 2-D PAGE confirms that the constitutive isozyme consists of only type 1 subunits while the major proliferation specific isozyme is composed of a type 1 subunit and a unique type 2 subunit. The type 1 and type 2 subunits differ by at least four charge units under native, nondenaturing conditions of electrophoresis but do not differ in molecular mass. The difference between the type 1 and type 2 subunits is covalent, as the difference in isoelectric point between the two subunits is stable to both 2% SDS and 8 M urea. The expression of TPI-2 does not correlate with the existence of the labile asparagine residues. Turnover studies indicate that the level of each subunit is regulated by differences in rates of synthesis rather than degradation but a precursor-product relationship between the subunits was not observed. Thus the mechanism for synthesis of TPI-2 must operate either during mRNA processing or nascent peptide synthesis and then only in cells from hominoid species.     

Evidence for a period of directional selection following gene duplication in a neurally expressed locus of triosephosphate isomerase

A striking correlation between neural expression and high net negative charge in some teleost isozymes led to the interesting, yet untested, suggestion that negative charge represents an adaptation (via natural selection) to the neural environment. We examine the evolution of the triosephosphate isomerase (TPI) gene family in fishes for periods of positive selection. Teleost fish express two TPI proteins, including a generally expressed, neutrally charged isozyme and a neurally expressed, negatively charged isozyme; more primitive fish express only a single, generally expressed TPI isozyme. The TPI gene phylogeny constructed from sequences isolated from two teleosts, a single acipenseriform, and other TPI sequences from the databases, supports a single gene duplication event early in the evolution of bony fishes. Comparisons between inferred ancestral TPI sequences indicate that the neural TPI isozyme evolved through a period of positive selection resulting in the biased accumulation of negatively charged amino acids. Further, the number of nucleotide changes required for the observed amino acid substitutions suggests that selection acted on the overall charge of the protein and not on specific key amino acids.     

Proteolytic modification of human phosphoglycerate kinase from lymphoblasts

During phytohemagglutinin and concanavalin A-induced transformation of human lymphocytes, phosphoglycerate kinase (PGK) exhibits new electrophoretic forms (pI = 8.5–8.9). Electrophoresis and electrofocusing showed that the new forms are not due to expression of the autosomally linked isozyme found in sperm (PGK-B; pI = 9.7). The multiple electrophoretic forms are the result of protease modification of the sex-linked PGK-A isozyme.     

The synthesis of a labile triosephosphate isomerase isozyme in human lymphoblasts and fibroblasts

Human peripheral lymphocytes contain a single electrophoretic form of triosephosphate isomerase (pI = 5.6). However, when induced to undergo blastogenesis by mitogens such as phytohemagglutinin or convanavalin A, a second isozyme (pI = 5.2) is also produced. This new isozyme is also found in human fibroblasts, but is present only in low concentrations in most other human tissues. The two isozymes were isolated from lymphocytes, lymphoblasts, and fibroblasts by isoelectric focusing, and their properties and the requirements for their synthesis were studied. The production of a new isozyme occurs concomitantly with blastogenesis and DNA synthesis, but when DNA synthesis is delayed by hydroxyurea, the appearance of the new isozyme is unaffected. The formation of the new isozyme is inhibited by actinomycin D and puromycin, and thus, appears to be dependent on both RNA and protein synthesis. Lymphocytes grown in the presence of [³H]leucine synthesize the new isozyme which is isotopically labeled, and pulse-chase experiments show that the two isozymes are not interconvertible. Although the two isozymes exhibit essentially identical catalytic properties, they differ markedly with regard to their stability, with the more acidic isozyme being much more labile. The lability of the more acidic isozyme may account for its low levels in most other tissues.     

Multicomponent synthesis of some new (1S,4S)-2,5-diazabicyclo[2.2.1]heptane-dithiocarbamates and their in vitro anti-proliferative activity against CaSki,MDA-MB-231 and SK-Lu-1 tumour cells as apoptosis inducing agents without necrosis

Identification of a new class of antitumor agent capable to induce apoptosis without triggering necrotic cell death event is challenging. The present communication describes the multicomponent synthesis of seven new (1S,4S)-2,5-diazabicyclo[2.2.1]heptane-dithiocarbamates and their in vitro antiproliferative activity on cervical cancer cell line (CaSki), breast cancer cell line (MDA-MB231), lung cancer cell line (SK-Lu-1) and human lymphocytes. Among the synthesized dithiocarbamates, compound 9e displayed significant antiproliferative activity without inducing any necrotic cell death (both on tumour cells and lymphocytes) and induced apoptosis in tumor cells by the caspase dependent apoptotic pathway. The compound 9e also exhibited greater tumor selectivity than human lymphocytes. In silico ADME predictions revealed that compound 9e has the potential to be developed as a drug candidate. Rapid chemical modifications of this lead are thus highly necessary for further investigation as a drug like safer antitumor candidate and also to achieve compounds with better activity profile.     

Interaction of triosephosphate isomerase from Staphylococcus aureus with plasminogen

Triosephosphate isomerase (TPI; EC 5. 3. 1. 1) displayed on the cell surface of Staphylococcus aureus acts as an adhesion molecule that binds to the capsule of Cryptococcus neoformans, a fungal pathogen. This study investigated the function of TPI on the cell surface of S. aureus and its interactions with biological substances such as fibronectin, fibrinogen, plasminogen, and thrombin were investigated. Binding of TPI to plasminogen was demonstrated by both surface plasmon resonance analysis and Far‐Western blotting. It is suggested that lysine residues contribute to this binding because the interaction was inhibited by ?‐aminocaproic acid. Activation of plasminogen to plasmin by staphylokinase or tissue plasminogen activator decreased in the presence of TPI, whereas TPI was degraded by plasmin. In other experiments, intact S. aureus cells had the ability to both increase and decrease plasminogen activation depending on the number of cells. Several molecules expressed on the surface of S. aureus were predicted to interact with plasminogen, resulting in its increased or decreased activation. These findings indicate that S. aureus sometimes localizes and sometimes disseminates in the host, depending on the molecules expressed under various conditions.     

Interpretation of triose phosphate isomerase isozymes in the cherimoya (Annona cherimola Mill.)

Detailed interpretation of triose phosphate isomerase (TPI) isozymes in seed plants has been restricted to only a few species. Three sets of TPI bands are regularly observed in the cherimoya(Annona cherimola), a primitive angiosperm. The slowest, set I, is expressed as one or three bands; the second-slowest set II, as one or two bands; and the fastest, set IV, as one or three bands. A faint set III, just cathodal to set IV, is detected rarely with overstaining. Set IV bands are expressed in macerated extracted pollen but not in pollen leachate. Dissociation-reassociation experiments reveal that the set II bands are heterodimers involving, in part, the enzymes involved in the set I bands. These data combined with those from full-sib progeny analysis lead us to propose a three-locus model to explain the TPI isozyme banding patterns in cherimoya. Sets I and IV consist of the allelic products of individual, single loci. Sets I and II occur in the cytoplasm. Set IV occurs in organelles. Set II isozymes are the intergenic heterodimers of the locus coding for set I and the locus coding for set III. Our results reported here are contrasted with the TPI isozyme patterns known for other vascular plants and suggest that the locus coding for set III may be a duplication of very ancient origin.This work was supported, in part, by funding from the Elvenia J. Slosson Endowment Fund.     

Characterization of two new electrophoretic variants of human triosephosphate isomerase: Stability,kinetic, and immunological properties

Two new electrophoretic variants of human triosephosphate isomerase (TPI) have been partially purified and characterized. The TPI Manchester variant, a cathodally migrating electrophoretic allozyme identified in an individual with the phenotype TPI 1-Manchester, is associated with a normal level of enzyme activity in erythrocytes and normal kinetic properties. It is very thermolabile at 55 and 57° C, although it is not uniquely sensitive to either guanidine-HCl or urea denaturation. The TPI Hiroshima-2 variant is an anodally migrating allozyme (the phenotype of proband is TPI 1-Hiroshima-2) with normal activity and kinetic properties and also normal stability characteristics. It is inactivated less by antisera raised against normal human TPI than either the normal or the Manchester allozyme. Dissociation-reassociation experiments utilizing these allozymes have confirmed that normal human red blood cell TPI isozymes are produced by a sequence of reactions (presumably deamidations) involving alternating subunits.Financial support was derived from Contract EY-77-C-02-2828 from the Department of Energy.     

Characterization of three electrophoretic variants of human erythrocyte triosephosphate isomerase found in Japanese

Three new electrophoretic variants of human erythrocyte triosephosphate isomerase (TPI) have been partially purified and compared with the normal isozyme with respect to stability, kinetics, and immunological properties. TPI 2HR1, an anodally migrating variant, was less stable than normal in guanidine denaturation and thermodenaturation tests, although it exhibited normal kinetic properties. The level of enzyme activity in erythrocytes from the proband with the phenotype TPI 1-2HR1 was about 60% of the normal mean. The variant allozyme TPI 2NG1, an anodally migrating allozyme associated with normal activity, was very thermolabile at 55 and 57°C. It was also much more labile than normal in stability tests in buffers at pH 5 and pH 10, although it exhibited normal kinetic and immunological properties. TPI 4NG1, a cathodally migrating variant associated with normal activity and normal kinetic as well as immunological properties, was more stable than normal in pH 5 buffer. Family studies demonstrated that the unique characteristics of these variants are genetically transmitted. In two-dimensional electrophoresis of purified isozymes the variant subunits were separated from the normal in the pI axis. However, there is no difference between the variants and the normal in the molecular weight axis, suggesting that the variants result from single amino acid substitutions.     

Evidence for duplication of the structural genes coding plastid and cytosolic isozymes of triose phosphate isomerase in diploid species of clarkia

Formal genetic analyses of the mode of inheritance of the multiple plastid and cytosolic isozymes of triose phosphate isomerase (TPI, EC 5.3.1.1) in annual diploid species of Clarkia (Onagraceae), native to California, suggest that each set of isozymes is specified by duplicate structural genes. In contrast, most diploid plant species possess one plastid and one cytosolic TPI isozyme each coded by a single locus. Linkage tests revealed that the two genes coding the plastid TPIs assort independently. Although the number of individuals sampled per species was small, the plastid isozymes were electrophoretically more variable than the cytosolic isozymes. The two gene duplications are the first reported that characterize an entire plant genus. Initial electrophoretic surveys of TPI in other genera of Onagraceae revealed that the duplication of the gene coding the plastid isozyme is apparently restricted to Clarkia, whereas that of the gene coding the cytosolic isozyme is present in most genera of the family. The separate phylogenetic distributions of the two duplications suggest that the processes that gave rise to them were unrelated.     

Changes in glycolytic enzyme levels and isozyme expression in human lymphocytes during blast transformation.

The levels of each of the glycolytic enzymes were observed to exhibit a parallel increase of 200 to 300% when human lymphocytes were stimulated to undergo blast transformation. A series of electrofocusing and electrophoretic studies was utilized to assess the isozyme distribution of the glycolytic enzymes during blastogenesis. Hexokinase (pI = 7.40), glucosephosphate isomerase (pI = 9.35), and enolase (pI = 8.30) existed as single electrophoretic components and were unchanged during blast transformation. Phosphoglycerate mutase was observed to exist as two isozymes (pI = 5.80 and 6.63), which were also unchanged by blastogenesis. Aldolase, which was present as two electrophoretic forms in lymphocytes (pI = 9.25 and 8.75), exhibited a shift in the relative content of each. In addition to the lactate dehydrogenase isozymes at pI 9.50 and 7.60 found in lymphocytes, lymphoblasts contained isozymes with pI values of 7.30, 7.05, and 5.85. Although glyceraldehyde 3-phosphate dehydrogenase was present as a single electrophoretic form (pI ? 8.0) in both lymphocytes and lymphoblasts, the association of the enzyme with actin produced electrophoretic artifacts with lower pI values. Phosphoglycerate kinase, which appeared as a single form in lymphocytes (pI = 9.00), was present as two isozymes (9.00 and 8.74) in lymphoblasts. Similarly, pyruvate kinase (pI = 8.73 and 8.50 in lymphocytes) exhibited additional isozymes (pyruvate kinase, pI = 7.60 and 5.85, and triosephosphate isomerase, pI = 5.20) as a result of cell transformation.     

Assignment of the genes for triose phosphate isomerase to chromosome 6 and tripeptidase-1 to chromosome 10 in Mus musculus by somatic cell hybridization

Evidence is presented for the assignment of the gene for triose phosphate isomerase to Mus musculus chromosome 6 and tripeptidase-1 to chromosome 10 by synteny testing and chromosome assignment in Chinese hamster X mouse somatic cell hybrid clones. Neither TPI nor TRIP-1 were expressed concordantly with any known isozyme markers in 45 hybrid clones (13 primary and 32 secondary). Karyotypic analysis of 21 clones showed that the expression of TPI and chromosome 6 were concordant in all cases as was expressed of TRIP-1 and chromosome 10. Both chromosomes were previously unmarked by isozymes.     

Characterization of invertebrate cell lines

Summary A reliable, simple method for the separation of four cellular isozymes utilizing celluloseacetate electrophoresis is described. Cell extracts of 16 cell lines were examined previously, utilizing starch gel electrophoretic techniques. These same cell extracts were retested for their isozyme phenotype using the cellulose-acetate electrophoretic system. These data indicate that the results of the two electrophoretic systems are comparable. Isozyme analyses of freshly prepared cell extracts of theAedes albopictus (ATC-15) andSpodoptera frugiperda (IPLB-SF-21AE) cell lines resulted in identical isozyme mobilities when compared with their respective stored counterparts. Since the cellulose-acetate electrophoretic system was able to separate cellular isozymes into characteristic patterns, distinctions between cell lines were made. The application of this technique for identification and characterization of invertebrate cell lines is discussed. This research was supported in part by the Rockefeller Foundation and U.S. Public Health Service Grant AI-13727.     

NF-kappaB activation for constitutive expression of VCAM-1 and ICAM-1 on B lymphocytes and plasma cells.

Cytokine stimulation can activate NF-kappaB that triggers inducible expression of E-selectin, VCAM-1 (Vascular Cell Adhesion Molecule-1) and ICAM-1 (Intercellular Cell Adhesion Molecule-1) in endothelial cells. In the previous study, we have shown that B lymphocytes and plasma cells can express E-selectin by constitutive activation of NF-kappaB. Here we show that human B lymphocytes and ARH-77 plasma cells expressed VCAM-1 and ICAM-1 in a cytokine dispensable mechanism. NF-kappaB antagonists could inhibit their expressions in ARH-77 cells. The activities of NF-kappaB for VCAM-1 and ICAM-1 promoters prior to cytokine stimulation were detected in ARH-77 cells using electrophoretic mobility shift assays. Again, NF-kappaB antagonists could abrogate these promoter activities. Taken together, our results demonstrate that NF-kappaB activation is the underlying molecular mechanism for constitutive expression of E-selectin, VCAM-1, and ICAM-1 on human B lymphocytes and plasma cells.     

6-Phosphogluconate Dehydrogenase (6PGD) Gene Duplication in Kalimeris (Asteraceae)

Abstract The genus Kalimeris with a diagnostic character of short or inconspicuous pappus consists of two sections, Asteromoea and Cordifolium. As a result of 6PGD isozyme analysis, sect, Asteromoea, including 2 × and poly-ploid taxa from 5 × to 8 ×, show similar cytosolic isozyme multiplicity and share a monomorphic locus. The data suggest that gene duplication of polyploid members was derived from a common ancestor. K. miqueliana, belonging to sect. Cordifolium. also possessed a gene duplication in 6PGD, though significant differences were detected in electrophoretic mobility between the sections. The occurrence of gene duplication in East Asian diploid Astereae leaves intact the validity of the allopolyploid-origin hypothesis of n= 9, which was rejected by Gottlieb (1981a) in American Astereae.     

Malate dehydrogenase isozymes in flax genotroph leaves: Differences in apparent molecular weight and charge between and within L and S

Ferguson plots demonstrated that corresponding malate dehydrogenase (MDH) isozymes of Durrant's L and S flax genotrophs differ in apparent molecular weight (MW) and also in net negative charge. The MW differences explain heritable differences in electrophoretic relative mobility (R _m) between corresponding L and S isozymes. The MW for each MDH isozyme was higher for L than for S and resulted in a slowerR _m for L. The net negative charge for each isozyme was higher for L than for S. MDH isozymes also differ in MW within L and S. MW was lower for isozymes in leaves from the bottom of the stem than in leaves from the top of the stem, particularly in L. Integration of information on the MDH isozyme system in the flax genotrophs and information on the peroxidase system suggests the possibility that common modifier loci may controlR _m in both enzymes.The financial assistance of the Natural Sciences and Engineering Research Council of Canada is acknowledged with thanks.