A single Gly114Arg mutation stabilizes the hexameric subunit assembly and changes the substrate specificity of halo-archaeal nucleoside diphosphate kinase

Nucleoside diphosphate kinase from extremely halophilic archaeon (HsNDK) requires above 2M NaCl concentration for in vitro refolding. Here an attempt was made to isolate mutations that allow HsNDK to refold in low salt media. Such a screening resulted in isolation of an HsNDK mutant, Gly114Arg, which efficiently refolded in the presence of 1M NaCl. This mutant, unlike the wild type enzyme, was expressed in Escherichia coli as an active form. The residue 114 is in close proximity to Glu155 of the neighboring subunit in the three dimensional hexameric structure of the HsNDK. It is thus possible that the attractive electrostatic interactions occur between Arg114 and Glu155 in the mutant HsNDK, stabilizing the hexameric subunit assembly.     

Residue 134 determines the dimer-tetramer assembly of nucleoside diphosphate kinase from moderately halophilic bacteria

Halomonas nucleoside diphosphate kinase (HaNDK) forms a dimeric assembly and Pseudomonas NDK (PaNDK) forms a tetrameric assembly. The mutation of Glu134 to Ala in HaNDK resulted in the conversion of the native dimeric structure to the tetramer assembly. Conversely, the mutation of Ala134 to Glu in PaNDK lead to the conversion from the tetramer to the dimer assembly, indicating that a single amino acid substitution at position 134 results in an alteration of the oligomeric structure of NDK. By modeling the structure of HaNDK and PaNDK based on the crystal structure of Myxococcus NDK, we showed that Glu134 exerts sufficient repulsive forces to disrupt the dimer-dimer interaction and prevent the formation of the tetramer.     

Interaction of hexa-His tag with acidic amino acids results in facilitated refolding of halophilic nucleoside diphosphate kinase

We have previously reported that amino-terminal extension sequence containing hexa-His facilitated refolding and assembly of hexameric nucleoside diphosphate kinase from extremely halophilic archaeon Halobacterium salinarum (NDK). In this study, we made various mutations in both the tag sequence and within NDK molecule. SerNDK, in which hexa-His was replaced with hexa-Ser, showed no facilitated folding. In addition, HisD58GD63G, in which both Asp58 and Asp63 in NDK were replaced with Gly, also showed no refolding enhancement. These results suggest that hexa-His in His-tag interact cooperatively with either Asp58 or Asp63 or both. Furthermore, G114D mutant, which formed a dimer in low salt solution, was strongly stabilized by His-tag to form a stable hexamer.     

Secondary and quaternary structural transition of the halophilic archaeon nucleoside diphosphate kinase under high- and low-salt conditions

Most halophilic enzymes from extremely halophilic archaea are denatured immediately after transfer from high-salt to low-salt medium. However, nucleoside diphosphate kinase (HsNDK) from the extremely halophilic archaeon Halobacterium salinarum seems to be exceptional, since the enzyme exhibited catalytic activity even under the low-salt condition. Here we show the mechanism how HsNDK is active under both high- and low-salt conditions that the HsNDK hexamer in high-salt medium dissociates into a dimer in the low-salt medium without denaturation. The observed change of the subunit structure was accompanied by a large decrease of alpha-helical content and lowered thermal sensitivity, yet keeping the conformations. This novel hexamer to dimer conversion under high- and low-salt conditions, respectively, seems to be the mechanism by which HsNDK is avoided from the irreversible denaturation.     

Structure of a halophilic nucleoside diphosphate kinase from Halobacterium salinarum

Nucleoside diphosphate kinase from the halophilic archaeon Halobacterium salinarum was crystallized in a free state and a substrate-bound form with CDP. The structures were solved to a resolution of 2.35 and 2.2A, respectively. Crystals with the apo-form were obtained with His6-tagged enzyme, whereas the untagged form was used for co-crystallization with the nucleotide. Crosslinking under different salt and pH conditions revealed a stronger oligomerization tendency for the tagged protein at low and high salt concentrations. The influence of the His6-tag on the halophilic nature of the enzyme is discussed on the basis of the observed structural properties.     

Metabolism of Histidine in Growing Rats at Various Dietary Protein Levels

The metabolic fate of the carbon skeleton of l-(U-¹⁴C)-histidine has been investigated in growing rats fed diets containing different percentages of protein calories (0, 5, 10, 15 and 30 PC%) at 410 kcal of metabolizable energy per 100 g diet.

The incorporation of ¹⁴C into body protein at 12 hr after injection of ¹⁴C-histidine was about 70% of the dose in rats fed 0 to 10 PC% diets, though the value was reduced in rats fed higher PC% diets. The expired ¹⁴CO₂ production was depressed in the low protein groups, and it showed an inverse pattern to that of ¹⁴C incorporation into body protein. Urinary excretion of ¹⁴C was about 20% of the dose in all dietary groups. The activities of hepatic histidase, urocanase and histidine-pyruvate aminotransferase were increased in the 30 PC% group.

These results indicate that the metabolic response of histidine to dietary protein changes around 10 PC%, where growth rate and body protein retention reached approximate plateaus.

The nutritional significance of the metabolism of histidine has been discussed and compared with that of leucine, alanine and serine reported previously.     

Hyperthermia suppresses the cytotoxicity of NK cells via down-regulation of perforin/granzyme B expression

Hyperthermia, which is used as an adjunctive therapy for cancer, is known to modulate the activity of natural killer (NK) cells in vitro, but its effect in vivo is unclear. In the present study, we used a whole body hyperthermia (WBH) device heated by infrared rays to evaluate the effect of WBH on mice models. We demonstrate here that wild type C57BL/6J mice exposed to 42 degrees C for 60min had reduced NK cell cytolytic activity against YAC-1 target cells as determined by cytolytic assay. This result was confirmed using Rag-2 knockout mice, which possess functional NK but not cytolytic T or NK-T cells. Moreover, WBH decreased the mRNA expression of perforin and granzyme B in spleens of mice. But the expression of TNF cytokines (Fas ligand and TRAIL) was unchanged. These data suggest that the suppression of NK cell activity induced by WBH could be mediated through the perforin/granzyme pathway.     

Toxoplasma gondii inhibits granzyme B-mediated apoptosis by the inhibition of granzyme B function in host cells

Host defense to the apicomplexan parasite Toxoplasma gondii is critically dependent on CD8⁺ T cells, whose effector functions include the induction of apoptosis in target cells following the secretion of granzyme proteases. Here we demonstrate that T. gondii induces resistance of host cells to apoptosis induced by recombinant granzyme B. Granzyme B induction of caspase-independent cytochrome c release was blocked in T. gondii-infected cells. Prevention of apoptosis could not be attributed to altered expression of the Bcl-2 family of apoptotic regulatory proteins, but was instead associated with reduced granzyme B-mediated, caspase-independent cleavage of procaspase 3 to the p20 form in T. gondii-infected cells, as well as reduced granzyme B-mediated cleavage of the artificial granzyme B substrate, GranToxiLux. The reduction in granzyme B proteolytic function in T. gondii-infected cells could not be attributed to altered granzyme B uptake or reduced trafficking of granzyme B to the cytosol, implying a T. gondii-mediated inhibition of granzyme B activity. Apoptosis and GranToxiLux cleavage were similarly inhibited in T. gondii-infected cells exposed to the natural killer-like cell line YT-1. The endogenous granzyme B inhibitor PI-9 was not up-regulated in infected cells. We believe these findings represent the first demonstration of granzyme B inhibition by a cellular pathogen and indicate a new modality for host cell protection by T. gondii that may contribute to parasite immune evasion.     

Enhancement of redirected target cell lysis by cytotoxic T lymphocytes in the presence of cytochalasin B

The cytochalasins are known secretogogues. Their function as such is examined in light of the granule exocytosis model for lymphocyte-mediated cytotoxicity. Cytochalasin B is found to enhance target cell lysis by cytotoxic T lymphocytes when antibody-coated polystyrene beads are used to bridge the cells. The pattern of lysis is found to be biphasic in its dependence on cytochalasin B. Secretion of the enzyme BLT-esterase from the effector cells parallels the cytochalasin concentration-dependent pattern of lysis. Cytochalasin D is also able to enhance lysis but at concentrations less than cytochalasin B. Cytochalasin B does not inhibit binding of breads to the effector cell. This is shown by the ability of fluorescent beads coated with antibody to bind with an appropriate specificity to cells. These studies indicate that cytochalasin B is not strictly inhibitory for the induction of target cell lysis but can enhance lymphocyte-mediated lysis at low drug concentrations. These results are compatible with the interpretation that target cell lysis is mediated through a secretion process from cytotoxic T lymphocytes.     

Modulation of perforin, granzyme A, and granzyme B in murine natural killer (NK), IL2 stimulated NK, and lymphokine-activated killer cells by alcohol consumption.

Alcohol consumption in mice suppresses the cytolytic activity of natural killer (NK) and lymphokine-activated killer (LAK) cells through unknown mechanisms. Herein, we found that alcohol consumption decreased target cell-induced release of granzyme A activity in freshly isolated splenic NK cells, in NK cells stimulated for 18 h with 1000 IU/ml of interleukin 2, and in LAK cells. The total activity and protein expression of granzymes A and B also were lower in these cells than in cells isolated from water-drinking mice. Interleukin 2 increased granzyme A protein expression independent of alcohol consumption; however, this increase was associated with decreased enzyme activity. In contrast, granzyme B protein expression and enzymatic activity increased in response to interleukin 2. Perforin activity and protein expression were reduced in LAK cells generated from alcohol-consuming mice. We conclude that the mechanism underlying the suppression of NK and LAK cytolytic activity by alcohol consumption involves the collective reduction of target-induced release, activity, and expression of perforin and granular proteases.     

An Ecto-Nucleotide Pyrophosphatase Is One of the Main Enzymes Involved in the Extracellular Metabolism of ATP in Rat C6 Glioma

Abstract : The presence of a nucleotide pyrophosphatase (EC 3.6.1.9) on the plasma membrane of rat C6 glioma has been demonstrated by analysis of the hydrolysis of ATP labeled in the base and in the α-and γ-phosphates. The enzyme degraded ATP into AMP and PP_i and, depending on the ATP concentration, accounted for ~50-75% of the extracellular degradation of ATP. The association of the enzyme with the plasma membrane was confirmed by ATP hydrolysis in the presence of a varying concentration of pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a membrane-impermeable inhibitor of the enzyme. PPADS concentration above 20 μ M abolished the degradation of ATP into AMP and PP_i. The nucleotide pyrophosphatase has an alkaline pH optimum and a K _m for ATP of 17 ± 5 μ M . The enzyme has a broad substrate specificity and hydrolyzes nucleoside triphosphates, nucleoside diphosphates, dinucleoside polyphosphates, and nucleoside monophosphate esters but is inhibited by nucleoside monophosphates, adenosine 3',5'-bisphosphate, and PPADS. The substrate specificity characterizes the enzyme as a nucleotide pyrophosphatase/phosphodiesterase I (PD-I). Immunoblotting and autoadenylylation identified the enzyme as a plasma cell differentiation antigen-related protein. Hydrolysis of ATP terminates the autophosphorylation of a nucleoside diphosphate kinase (NDPK/nm23) detected in the conditioned medium of C6 cultures. A function of the pyrophosphatase/PD-I and NDPK in the purinergic and pyrimidinergic signal transduction in C6 is discussed.     

Distribution of the 7S Proteins in Soybean Globulins by Gel Filtration with Sephadex G-200

The level of isocitrate lyase, an enzyme of glyoxylate cycle, in Candida tropicalis was enhanced at the later period of growth when the yeast was cultivated in a semisynthetic glucose medium. On the other hand, such increase in the enzyme activity was not observed in C. lipolytica grown under the same conditions. In the case of C. tropicalis, high concentrations of glucose remaining in the medium permitted the increase in the enzyme activity and the addition of ethanol, one of the major products from glucose, to the glucose medium did not stimulate the enzyme formation, indicating that the enhanced enzyme level in the yeast was not merely attributable to the release from the repression by glucose or to the induction by ethanol. Biotin, one of the growth-stimulating factors for C. tropicalis, affected markedly the level of isocitrate lyase. That is, the supplementation of biotin to the synthetic glucose medium inhibited completely the increase in the enzyme activity, and reversely the absence of biotin stimulated the enzyme formation in the glucose-assimilating cells. Thiamine, another growth-stimulating factor for C. tropicalis, did not show any effect on the level of isocitrate lyase in the yeast. The level of isocitrate lyase in C. lipolytica growing on glucose was not affected by biotin added exogenously.     

Local coexpression domains in the genome of rice show no microsynteny with Arabidopsis domains

Plant responses to abiotic stress are determined both by the severity of the stress as well as the metabolic status of the plant. Abscisic acid (ABA) is a key component in integrating these various signals and controlling downstream stress responses. By screening for plants with decreased RD29A:LUC expression, we isolated two alleles, glutamate:glyoxylate transferase1-1 (ggt1-1) and ggt1-2, of a mutant with altered ABA sensitivity. In addition to reduced ABA induction of RD29A, ggt1-1 was altered in ABA and stress regulation of Δ ¹ -pyrroline-5-carboxylate synthase, proline dehydrogenase and 9-cis-epoxycarotenoid dioxygenase 3, which encode enzymes involved in Pro and ABA metabolsim, respectively. ggt1-1 also had altered ABA and Pro contents after stress or ABA treatments while root growth and leaf water loss were relatively unaffected. A light-dependent increase in H₂O₂ accumulation was observed in ggt1-1 consistent with the previously characterized role of GGT1 in photorespiration. Treatment with exogenous H₂O₂, as well as analysis of a mutant in nucleoside diphosphate kinase 2 which also had increased H₂O₂ content but is not involved in photorespiration or amino acid metabolism, demonstrated that the greater ABA stimulation of Pro accumulation in these mutants was caused by altered H₂O₂ content as opposed to other metabolic changes. The results suggest that metabolic changes that alter H₂O₂ levels can affect both ABA accumulation and ABA sensitivity. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Changes in the activity of enzymes involved in purine "salvage" and nucleic acid degradation during the growth of Catharanthus roseus cells in suspension culture

Changes during growth in the activity of several enzymes involved in purine "salvage", adenine phosphoribosyltransferase (EC 2.4.2.7), guanine phosphoribosyl-transferase (EC 2.4.2.8), hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) and adenosine kinase (EC 2.7.1.20), the enzymes which catalyze the conversion of nucleoside monophosphate to triphosphate, nucleoside monophosphate kinase (EC 2.7.4.4) and nucleoside diphosphate kinase (EC 2.7.4.6), and several degradation enzymes, deoxyribonucleae(s), ribonuclease(s). phosphatase(s), nucleosidase (EC 3.2.2.1), 3'-nucleotidase (EC 3.1.3.6) and 5'-nucleotidase (EC 3.1.3.5) were examined in cells of Catharanthus roseus (L.) G. Don cultured in suspension. In addition, the incorporation of [8-¹⁴C] adenine, [8-¹⁴C] adenine, [8-¹⁴C]hypoxanthine. [8-¹⁴C] adenosine and [8-¹⁴C]inosine into nucleotides and nucleic acids was also determined using intact cells.
The activities of all purine "salvage" enzymes examined and those of nucleoside monophosphate and diphosphate kinases increased rapidly during the lag phase and decreased during the following cell division and cell expansion phases. The rate of incorporation of adenine, guanine, hypoxanthine, and adenosine into nucleotides and nucleic acids was higher in the lag phase cells than during the following three phases. The highest rate of [8-¹⁴C]inosine incorporation was observed in the stationary phase cells. The activity of all degradation enzymes examined decreased when the stationary phase cells were transferred to a new medium.
These results indicated that the increased activity of purine "salvage" enzymes observed in the lag phase cells may contribute to an active purine "salvage" which is required to initiate a subsequent cell division.     

Nucleoside diphosphate kinase-1 regulates hyphal development via the transcriptional regulation of catalase in Neurospora crassa

A nucleoside diphosphate kinase-1-disrupted (ndk-1^RIP-1) mutant was observed to be defective in aerial hyphal and conidial development. In this study, two types of hyphae, fine and thick, were observed in wild-type (Wt) strains. However, only fine-type hyphae were observed in the ndk-1^RIP-1 mutants. The ndk-1^RIP-1 mutants were stimulated by oxidative stress and constitutively expressed an antioxidant enzyme catalase (CAT)-3. Furthermore the ndk-1^RIP-1 mutants could form thick hyphae by oxidative stress and a disruption of cat-3. These results suggest that the loss of thick hyphae in the ndk-1^RIP-1 mutants may be caused by the over-expression of cat-3.     

Evolution of acid phosphatase-1 in the genus drosophila as estimated by subunit hybridization. Interspecific tests

Summary The dimeric enzyme, acid phosphatase-1, was partially purified from eleven species of the genus Drosophila. Dissociated subunits were mixed and allowed to reassociate in forty-one interspecific combinations. In each so-called quantitative subunit hybridization test, the relative activities of the heterospecific and the two homospecific enzymes were determined by densitometry. In 34 of the 41 tests significant differences between observed and expected homospecific: heterospecific enzyme activity ratios were detected. The differences ranged from a four-fold excess of the heterospecific enzyme to over a six-fold excess of the homospecific enzymes. In order to measure the enzyme activities on a protein basis, fifteen heterospecific enzymes were purified and used as antigens in CRM tests. The antisera were diluted such that only the homologous subunit in the heterospecific enzyme complexed the acid phosphatase antibodies. The results from each CRM test show that the heterospecific enzymes is only one-half as antigenic as the homologous homospecific enzyme, when the two are adjusted to equal catalytic activities. Thus, the differences between observed and expected levels of acid phosphatase activity measured by the quantative subunit hybridization technique apparently reflect differences in the relative amounts of protein which form during subunit reassociation. The technique, then, appears to detect differences in acid phosphatase subunit affinities.The data either taken directly from the 41 interspecific tests or in terms of the average difference between each two species in third species tests were used to construct phenograms. The species relationships depicted in both phenograms were very different from their actual phylogenetic relationships. This method, then, is not useful as an evolutionary metric. The differences between observed and expected heterospecific:homospecific enzyme ratios may be due to a relatively large number of amino acid substitutions if acid phosphatase subunits pair isologously.     

Role of perforin, granzymes and the proliferative state of the target cells in apoptosis and necrosis mediated by bispecific-antibody-activated cytotoxic T cells

 Bispecific monoclonal antibodies (bi-mAb), directed against a tumor-associated antigen and the CD3 or CD28 antigen on T lymphocytes, induce activation of resting T lymphocytes and target-specific tumor cell lysis. We now show that both necrosis and apoptosis contribute to T-cell-mediated tumor cell destruction. Even though T cells up-regulate FAS/APO-1 expression upon bi-mAb stimulation, FAS/APO-1-mediated apoptosis does not contribute to bi-mAb-mediated destruction of Hodgkin’s cells. CD8⁺ lymphocytes were the most potent effectors of bi-mAb-mediated cytotoxicity and had the highest levels of mRNA coding for perforin and granzyme A and B. Ca²⁺-complexing agents, which abrogate perforin activity, led to decreased levels of necrosis, while inhibition of granzyme activity in effector or target cells had a similar effect on apoptosis. Granzyme-mediated apoptosis critically dependent on the proliferative state of the target cells, while perforin-induced necrosis was not cell-cycle-dependent. Our results underline the importance of the expression levels of perforin and granzymes in the effector T cells and of the proliferative state of the target cells in bi-mAb-mediated apoptosis and necrosis of tumor cells. Received: 5 December 1996 / Accepted: 16 January 1997     

The human perforin gene is a direct target of STAT4 activated by IL-12 in NK cells

IL-12 activates STAT4 by inducing tyrosine phosphorylation, homo-dimerization, and nuclear translocation in NK cells and thereby stimulates proliferation and activation of these cells. The pore-forming protein perforin is a key effector protein for NK cell- and cytotoxic T lymphocyte-mediated cytolysis. Here we demonstrate that IL-12 induces the expression of the perforin gene in human NK cell line, NKL. Electrophoretic mobility shift assays using a probe containing two putative STAT-binding sequences located at -1085 and -1059 in the human perforin gene showed that STAT4 or STAT5 activated by IL-12 or IL-2, respectively, in NKL cells binds this region. Further analyses using various probes with or without mutated STAT-binding sequences showed that, although either of the two tandem STAT-binding sequences binds STAT4 weakly, the presence of both is required for significant binding of activated STAT4 and for formation of the STAT4-DNA-binding complex with lower electrophoretic mobility. Furthermore, mutation of either of the tandem STAT-binding sequences abolished the IL-12-induced activation of the perforin gene promoter in reporter gene assays. These results indicate that the IL-12-induced expression of the perforin gene in NK cells is directly regulated by STAT4, which binds, most likely as a homo-tetramer, to the tandem STAT-binding sequences in the perforin gene promoter.     

Increasing the refolding efficiency in vitro by site-directed mutagenesis of Cys383 in rat procarboxypeptidase B

This study examines a novel method to reduce the probability of disulfide mismatches during the refolding process by the replacement of cysteines within a protein. Specifically, Cys383 of recombinant rat procarboxypeptidase B was replaced by other amino acids to increase the refolding efficiency in vitro. Mutants C383G, C383A and C383S could refold successfully, but mutants C383R, C383E, C383L and C383Y failed to refold correctly. Compared with wild type, the refolding efficiencies of mutants C383G and C383A were enhanced. The Cys383 mutations changed some of the properties of rat carboxypeptidase B. Mutants C383G, C383A had higher k_cat/K_m values which indicated increased catalytic abilities. And both had higher thermal stability. pH had different effects on the activities and stabilities of the mutant and wild type proteins. The studies suggested that mutating Cys383 of rat procarboxypeptidase B could improve the renaturation process by increasing the refolding efficiency. This new method could be taken as a new attempt to improve the refolding efficiency of other recombinant proteins containing disulfide bonds that are expressed as inclusion bodies. While the results also claimed that the potential effects of the substituted amino acid on the protein itself should be seriously considered in addition to its ability to reduce the probability of disulfide mismatches.