M. Antonieta Valenzuela

Identification and subcellular localization of two isoenzymes of apyrase from Solanum tuberosum

Abstract Two forms of ATP-diphosphohydrolase were identified in Solanum tuberosum tuber var. Ultimus. Their hydrolytic activity ratios (ATPase/ADPase) were over 10 for form A and 1 for form B. In the potato tuber homogenate the hydrolytic activity ratio is 3.0, as a result of contributions of the two forms of apyrase. These two apyrases (A and B) were partially separated and the possibility that they are produced as an artifact by partial proteolysis or subunit aggregation was excluded. The subcellular localization of the Ultimus isoapyrases was studied by differential centrifugation. These enzymes are localized in distinct compartments. The high ratio enzyme (A) lies mainly in the soluble fraction, while the low ratio apyrase (B) is principally bound to membranes. The two isoapyrases differ greatly in their kinetic properties and p I , but only slightly in M r . Both enzymes immunocross-react with antiapyrase Desiree, which is important for isoenzyme detection by the immunowestern blot. This is the first example of two isoenzymes of apyrase in the same variety of S. tuberosum .

Purification and characterization of two isoapyrases from Solanum tuberosum var. ultimus

Abstract Two isoenzymes of ATP-diphosphohydrolase (apyrase) were extracted and purified from S. tuberosum var. Ultimus. Their hydrolytic activity ratios (ATPase/ADPase) were 1.0 (apyrase B) and ca 15.0 (apyrase A). They were characterized and compared with apyrases of other varieties of S. tuberosum . Ultimus apyrases, like the other apyrases, did not hydrolyse esteric bonds but only pyrophosphate bonds of organic and inorganic compounds. The optimum pH of all the studied hydrolytic activities of the Ultimus apyrases A and B was 6, except for the ADPase of enzyme A which was 8. Both enzymes require bivalent metal ions for catalytic activity. The activation order for both Ultimus enzymes was: Ca 2+ >Mn 2+ > Mg 2+ >Co 2+ >Zn 2+ . Chemical modification of tryptophan, tyrosine, arginine and carboxylic residues decreased all enzymic activities of both apyrases. The modification of histidine residues reduced the ATPase and ADPase activities of the low ratio apyrase and the ATPase of the high ratio enzyme but did not affect its ADPase activity. Neither of the Ultimus apyrases showed the participation of -SH groups in the active site. The pI values obtained were: 5.45 for apyrase B and 6.56 for apyrase A. The absorption and the fluorescence spectra of the Ultimus isoenzymes were coincident. The amino acid composition of both isoenzymes is very similar, the number of histidines being the most remarkable difference. The amino acid composition of both isoenzymes does not explain the difference of one pH unit in the isoelectric point between the Ultimus enzymes A and B.

TIMPs and MMPs expression in CSF from patients with TSP/HAM

The tropical spastic paraparesis or human T-cell lymphotropic virus associated myelopathy (TSP/HAM), has been related with an overexpression of matrix metalloproteinases (MMPs), especially MMP-9. Initial studies of reverse zymography with cerebrospinal fluid (CSF) from TSP/HAM patients, and controls showed the presence of TIMPs, endogenous MMP inhibitors. We determined in CSF the levels of TIMPs by immunoanalysis in 25 patients with TSP/HAM, and compared with two groups: controls and patients with acute and subacute inflammatory neurological diseases. We found that TIMP-2, TIMP-3 and TIMP-4 levels were significantly higher than in controls in both TSP/HAM and inflammatory patients, while TIMP-1 was increased only in the inflammatory group. Levels of MMP-3 and MMP-9 from the two groups of patients showed a significant upregulation in CSF. In the CSF of around the 70% of TSP-HAM and inflammatory patients the presence MMP-9 was detected by zymography, but not in controls. MMP-2 was only overexpressed in the acute inflammatory group. The active form of MMP-2 was observed in both groups of patients with a similar high frequency (60%). MMPs overexpressions are independent of the evolution time of the disease in TSP/HAM. The chronic overexpression of these extracelullar matrix proteins detected in CSF of TSP/HAM should be indirectly produced by secreted viral proteins being responsible for the progression of this disease, accounting for the observed differences with acute inflammatory patients. Our results support the existence of an imbalance between MMPs and their endogenous tissue inhibitors, which could be a pathogenic factor in the chronicity of TSP/HAM.

Structural studies of two apyrases from Solanum tuberosum

Abstract The proportion of acid and basic amino acid residues obtained for two homogeneous isoenzymes of apyrase isolated from different clonal varieties of Solanum tuberosum (Pimpernel and Desiree) was essentially the same. This does not agree with the difference in pI values observed. Treatment with asparaginase and glutaminase caused partial inactivation of both enzyme activities in both isoenzymes, and pI values were changed, but not equalized. The differences in pI values of the native isoenzymes may still be attributed to different proportions of glutamine and asparagine in the primary structure. Leucine is the amino-terminal residue in both isoenzymes. Both have two disulphide bridges and one buried sulphydryl group which is not essential for enzyme activity. Differences in pI values should thus be attributed to factors other than amino acid composition.

Identification of an apyrase activating protein and of calmodulin in Solanum tuberosum

Abstract Three types of effector proteins have been isolated from a partially purified protein preparation of potato tuber. One of the proteins is a typical calmodulin which has no effect on apyrase. The two other proteins modulate ATPase and ADPase activities; one of them with an activating and the other with an inhibitory effect on apyrase. Calmodulin from potato tuber purified to homogeneity had a M r of 17 500 and an isoelectric point of 4.4. Although the apyrase activating protein is not a pure fraction it differs from calmodulin because unlike this protein it is independent of calcium and does not activate cyclic nucleotide phosphodiesterase from bovine heart. Treatment of the activating protein with tetranitromethane reduces its effect on apyrase, while no change was detected upon treatment with bisdithionitrobenzoic acid.

Microtubule proteins and their post-translational forms in the cerebrospinal fluid of patients with paraparesis associated with HTLV-I infection and in SH-SY5Y cells: An in vitro model of HTLV-I-induced disease

HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is characterized by axonal degeneration of the corticospinal tracts. The specific requirements for transport of proteins and organelles to the distal part of the long axon are crucial in the corticospinal tracts. Microtubule dysfunction could be involved in this disease, configuring an axonal transport disease. We measured tubulin and its post-translational modified forms (acetylated and tyrosinated) in CSF of patients and controls, as well as tau and its phosphorylated forms. There were no significant differences in the contents of tubulin and acetyl-tubulin between patients and controls; tyrosyl-tubulin was not detected. In HAM/TSP, tau levels were significantly reduced, while the ratio of pT181/total tau was higher in patients than in controls, this being completely different from what is reported in other neurodegenerative diseases. Phosphorylation at T181 was also confirmed by Mass Spectrometry analysis. Western Blotting with monospecific polyclonal antibodies against pS199, pT205, pT231, pS262, pS356, pS396, pS404 and pS422 did not show differences in phosphorylation in these residues between patients and controls. Treating human SH-SY5Y neuroblastoma cells, a well-known in vitro neurite retraction model, with culture supernatant of MT-2 cells (HTLV-I infected cell line that secretes the viral Tax protein) we observed neurite retraction and an increase in tau phosphorylation at T181. A disruption of normal phosphorylation of tau protein in T181 could result in its dysfunction, contributing to axonal damage.

ATP-DIPHOSPHOPHYDROLASE ACTIVITY IN RAT HEART TISSUE

Extracellular nucleotides interact with specific receptors on the cell surface and are locally metabolized by ecto‐nucleotidases. Biochemical characterization of the ATPase and ADPase activities detected in rat heart sarcolemma, under conditions where mitochondrial ATPase and adenylate kinase were blocked, supports our proposal that both activities correspond to a single enzyme, known as ATP‐diphosphohydrolase or apyrase. The physiological function of this enzyme could be dephosphorylation of the nucleotides present in the interstitial heart compartment acting together with 5′‐nucleotidase. Both hydrolytic activities have similarities in: sarcolemma localization, bivalent metal ion dependence, optimum pH, effect of several amino acid residue modifiers, competitive inhibition of nucleotide analogs, and broad nucleoside di‐and triphosphate specificity. The ATPase activity could not be separated from the ADPase either through isoelectrofocusing or electrophoresis under acid conditions.

Nitric oxide synthase activity in brain tissues from llama fetuses submitted to hypoxemia.

The fetal llama (Lama glama; a species adapted to live in chronic hypoxia in the highlands of the Andes) did not increase cerebral blood flow and reduce the brain oxygen uptake during hypoxemia. Although nitric oxide (NO) is a normal mediator in the regulation of vascular tone and synaptic transmission, NO overproduction by hypoxemia could produce neuronal damage. We hypothesized that nitric oxide synthase (NOS) activity is either maintained or reduced in the central nervous system of the llama fetuses submitted to chronic hypoxemia. Approximately 85% of the Ca(2+)-dependent NOS activity was soluble, at least 12% was associated with the mitochondrial fraction, and less than 5% remains associated with microsomes. To understand the role of NO in chronic hypoxemia, we determined the effect of 24-h hypoxemia on NOS activity in the central nervous system. No changes in activity or the subcellular distribution of NOS activity in brain tissues after hypoxemia were found. We proposed that the lack of changes in NOS activity in the llama under hypoxemia could be a cytoprotective mechanism inherent to the llama, against possible toxic effects of NO.

ATP Diphosphohydrolase from Schistosoma mansoni Belongs to a New Family of Apyrases

ATP diphosphohydrolase from S. mansoni tegument was characterized as a 63-kDa protein with two isoforms possessing different isoelectric points. Antibodies against potato apyrase and Toxoplasma gondii nucleoside triphosphatase react with the 63-kDa protein, thus revealing the presence of common epitopes and suggesting that S. mansoni ATP diphosphohydrolase belongs to the family of apyrases. This is a new family of nucleotide-splitting enzymes so far unnoticed which became evident after sequencing 59 amino acids from potato apyrase. The novel potato apyrase sequences obtained have considerable homologies to eight sequences recently deposited in the data bank, including that of T. gondii nucleoside triphosphatase. The new family of apyrases contain a conserved motif (I/V)(V/M/I)(I/L/F/C)DAGS(S/T) near the amino terminal and three other regions showing four to six conserved amino acids.

Tax and Semaphorin 4D Released from Lymphocytes Infected with Human Lymphotropic Virus Type 1 and Their Effect on Neurite Growth

Human lymphotropic virus type 1 (HTLV-1) is a retrovirus causing HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a neurodegenerative central nervous system (CNS) axonopathy. This virus mainly infects CD4(+) T lymphocytes without evidence of neuronal infection. Viral Tax, secreted from infected lymphocytes infiltrated in the CNS, is proposed to alter intracellular pathways related to axonal cytoskeleton dynamics, producing neurological damage. Previous reports showed a higher proteolytic release of soluble Semaphorin 4D (sSEMA-4D) from CD4(+) T cells infected with HTLV-1. Soluble SEMA-4D binds to its receptor Plexin-B1, activating axonal growth collapse pathways in the CNS. In the current study, an increase was found in both SEMA-4D in CD4(+) T cells and sSEMA-4D released to the culture medium of peripheral blood mononuclear cells (PBMCs) from HAM/TSP patients compared to asymptomatic carriers and healthy donors. After a 16-h culture, infected PBMCs showed significantly higher levels of CRMP-2 phosphorylated at Ser(522). The effect was blocked either with anti-Tax or anti-SEMA-4D antibodies. The interaction of Tax and sSEMA-4D was found in secreted medium of PBMCs in patients, which might be associated with a leading role of Tax with the SEMA-4D-Plexin-B1 signaling pathway. In infected PBMCs, the migratory response after transwell assay showed that sSEMA-4D responding cells were CD4(+)Tax(+) T cells with a high CRMP-2 pSer(522) content. In the present study, the participation of Tax-sSEMA-4D in the reduction in neurite growth in PC12 cells produced by MT2 (HTLV-1-infected cell line) culture medium was observed. These results lead to the participation of plexins in the reported effects of infected lymphocytes on neuronal cells.