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The ITER neutral beam injection is based on the acceleration and neutralization of negative deuterium ions. The target performance for the ITER beam source is to accelerate to 1 MeV a 40 A D− beam, with a current density of 200 A m−2, with pulse lengths of ≥1000 s. It was found that in long pulse operation the negative ion yield from the filamented Kamaboko III ion source (a model of ITER ion source) degrades in comparison with short pulse operation

In this study we provide evidence that the protein kinase C (PKC)-straight theta isoenzyme is recruited on to the mitotic spindle in dividing murine erythroleukaemia (MEL) cells and associates specifically with centrosome and kinetochore structures. None of the other PKC isoenzymes (-alpha, -delta, -epsilon, -mu and -zeta) expressed by MEL cells shows this localization on the mitotic spindle. An identical subcellular distribution of PKC-straight theta is also observed in dividing murine P3 myeloma cells and human LAN-5 neuroblastoma cells, indicating that this PKC isoenzyme interacts with the mitotic apparatus in mammalian cells. In phorbol-ester-treated non-growing MEL cells, a rapid change in the intracellular distribution of PKC-straight theta occurs. Under these conditions, PKC-straight theta is translocated from the nuclear to the cytosolic cell compartment, an event that is accompanied by phosphorylation of the PKC-straight theta molecule and is followed by its down-regulation. The recovery of cell growth capacity results in the concomitant reappearance of PKC-straight theta. Furthermore, when MEL cells acquire the differentiated non-growing phenotype, the level of PKC-straight theta is reduced to less than 5%, suggesting that this PKC isoenzyme is no longer required. We propose that, unlike other members of the PKC family, PKC-straight theta may play a role in cell proliferation.

This work was undertaken to establish the forms of the calpain inhibitor, calpastatin, expressed in the brain tissue. Five cDNA clones were obtained and the corresponding amino acid sequences were deduced. Three of these proteins contain an N-terminal domain (domain L) and four inhibitory repeats typical of the calpastatin molecule. The other two are truncated forms, containing the domain L, free or associated with a single inhibitory repeat. Other differences, due to exon skipping, produce calpastatin forms with different susceptibility to posttranslational modifications. The more represented mRNA form corresponds to a calpastatin molecule containing the four inhibitory domains. These results may be useful to understand the involvement of calpain in the onset of acute and degenerative disorders of the central nervous system.

In mature red cells of rats from Milan normal (MNS) and hypertensive strains (MHS), the soluble Ca2+ dependent neutral proteinase (calpain) is present in similar amounts with identical Mr of 110 kDa and a dimeric structure composed of two unequal subunits of Mr of 84 and 26 kDa. Conversely, the amount of the endogenous inhibitor is now confirmed by analysis of the specific activity to be approximately 10 times less in red cells of MHS rats. The inhibitor is present in red cells of both strains in three different oligomeric forms of Mr of 240, 120 and 64 kDa. This last molecular species corresponds to the single basic constituent subunit which is the reacting inhibitor form. The apparent equilibrium between the three oligomeric structures is Ca2+-dependent. The high (0.1 mM) Ca2+ requirement for the activity of calpain from erythrocytes of both strains is reduced to 1-5 microM in the presence of plasma membrane phospholipids. Activation of the enzyme in these conditions is prevented by the natural inhibitor. These results strongly support and further emphasize the hypothesis that the structural and functional abnormalities in MHS rats red cells result from an impairment in the modulation of intracellular calpain activity by interaction with its endogenous inhibitor.

Fructose 1,6-bisphosphatase has been isolated in homogeneous form from rat liver by a simple and convenient procedure, including adsorption on carboxymethyl cellulose and substrate elution. It is a tetrameric protein, with molecular weight of approximately 140,000. The amino acid composition shows some similarity to that of the rabbit liver enzyme, with a blocked NH 2 -terminus and leucine, rather than alanine, as the COOH-terminal residue. The purified enzyme contains no tryptophan. It requires Mg 2+ or Mn 2+ and is activated by NH 4 + , which also decreases the affinity for both substrate and divalent cations. In the absence of chelating agents, the pH optimum is 8.0, but this is shifted to 7.0, 7.2, and 7.5 on addition of EDTA, histidine, and citrate, respectively. Ca 2+ is a potent inhibitor and appears to compete for the Mg 2+ -binding site. The rat liver enzyme is inhibited by AMP in a cooperative manner, with a Hill coefficient of about 2. Saturation is reached when approximately 2 mol of AMP are bound per mole of enzyme, but no AMP binding is observed in the absence of the substrate. Antibody to the purified enzyme, produced in the rabbit, reacts with fructose 1,6-bisphosphatase in rat liver and rat kidney with equal affinity. It also reacts with rat muscle fructose 1,6-bisphosphatase, with 1/100th the affinity.

Rat liver fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) contains 12 binding sites for Zn2+ per molecule, or 3 per subunit, as determined by gel filtration and by precipitation of an insoluble Zn2+-enzyme complex. The first set of sites binds Zn2+ with very high affinity, and the binding constant for these sites could not be determined. The average values of the dissociation constants for the second and third sets of sites were approximately 0.4 and 1.5 muM, respectively. The third set of sites, having lowest affinity, appears to be identical to the binding sites for the activating cation, Mg2+, and the binding of Zn2+ to this set of sites is prevented by the addition of Mg2+. Binding of the first 4 equivalents of Zn2+ yields an enzyme of intermediate activity, while the binding of 8 equivalent results in almost complete inhibition of catalytic activity. Thus Zn2+ appears to function as both an activator and a negative allosteric regulator of fructose-1,6-bisphosphatase activity.

FX is a red cell NADP(H)-binding protein that has been well defined biochemically and immunologically but whose function is still unknown. Preliminary data indicated that the levels of this protein are significantly increased in hemizygotes, heterozygotes, and homozygotes for the G6PD Mediterranean mutant, thus raising the question of whether or not the individual variation in FX levels is more or less directly influenced by X-linked genes. The present study, based on a large series of population and family data collected in Sardinia, confirms unequivocally the above mentioned interaction, but shows at the same time that the variances in FX levels “between sibships” are 2–3 times larger than those “within sibships,” when the analysis is done separately for the G6PD-normal or the G6PD-deficient sibs. From the comparison of the interclass and intraclass correlation coefficients, it appears that about 60% of the total variation of FX is of genetic origin. Moreover, the FX levels of children, analyzed in a pairwise manner, were found to be more positively correlated with those of their fathers (r = 0.39) than with those of their maternal grandfathers (0.20). This latter finding obviously favors the conclusion that “autosomal”; rather than “X-linked” genes are involved in the determination of the FX levels.

Fructose 1,6-diphosphatase (FDPase) has been purified about 10-fold from extracts of Escherichia coli B. The properties of the enzyme are similar to those reported for FDPases from other sources: high specificity for the substrate fructose 1,6-diphosphate, a low value of K m (about 10 −5 M ), requirement for a metal ion, and sensitivity to inhibition by low concentrations of adenosine-5′-monophosphate. The enzyme differs from most other FDPases in having a pH optimum in the physiological pH range.

Fructose-1, 6-diphosphatase purified from rabbit liver has a molecular weight of 127,000. The amino acid composition was determined. The protein contains about 20 sulfhydryl groups, nearly all of which can be titrated with p -hydroxymercuribenzoate (PHMB) in the absence of urea. Titration of 2 sulfhydryl groups with p -hydroxymercuribenzoate produces a 2-fold increase in enzyme activity; a similar effect is produced by iodoacetamide. The effect of PHMB is reversed by cysteine and abolished by digestion with papain. The results are compared with those obtained by activation with fluorodinitrobenzene.

At neutral pH, Zn2+ is a potent and specific inhibitor of rat liver fructose 1,6-bisphosphatase (EC 3.1.3.11; D-fructose-1,6-bisphosphate 1-phosphohydrolase). Inhibition by Zn2+ is uncompetitive with respect to the activating cations Mg2+ and Mn2+, and the kinetic data suggest that the enzyme possesses a distinct high-affinity binding site for Zn2+, with Ki of approximately 0.3 muM. At higher concentrations (about 10(-5) M) Zn2+, and to a lesser extent Co2+, function as activating cations. Binding studies show that the enzyme binds two equivalents of Zn2+ per subunit; one equivalent is partially displaced by Mg2+ and is presumably bound to the site for activating cations. A second equivalent binds to the high-affinity site, presumably identical to the inhibitory site. The results suggest that Zn2+ functions as an allosteric regulator, and that the commonly observed activation of fructose 1,6-bisphosphatase at neutral pH by EDTA, histidine, and other chelators is due to removal of endogenous Zn2+ by these agents.

Ca2+-dependent neutral proteinase purifies from human erythrocytes as an inactive proenzyme, that can be converted in an active low Ca2+ requiring form either by high concentrations of Ca2+ (0.1-1 mM) in the absence of the substrate, or by low concentrations of Ca2+ (1-5 microM) in the presence of digestible substrates. Activation requires dissociation to constituent inactive proenzyme subunits which are then converted to the active proteinase species still retaining their monomeric structure. The activation process produced by high Ca2+ concentrations is controlled by the endogenous inhibitor which also dissociates into constituent subunits in order to exert its inhibitory effect. An additional regulation of the activated proteinase involves an autoproteolytic process, Ca2+ and substrate dependent, producing enzyme inactivation.

These and earlier findings suggest that certain neutrophil biochemical responses induced by external stimuli are mediated by the reorganization of the cytoskeletal-membrane interactions. This rearrangement of the intracellular architecture appears to result from a coordinated and integrated operation of two enzymes, namely PKC and calpain. This conclusion is schematically represented in a comprehensive model, discussed in the introduction and derived from a number of experimental observations. In focusing the importance of cytoskeletal reorganization, signal-directed proteolysis, induced by specific PKC-mediated phosphorylation, appears to play a fundamental role in cell functions such as positioning and fusion of organelles with plasma membranes and possibly locomotion and transport.

In addition to other proteinases human neutrophils contain two non granular neutral endopeptidases: a serine proteinase and a cysteine Ca2+ dependent proteinase named calpain. Serine proteinase localized in association with the cytoskeleton-membrane proteins, apparently exerts a dual role: it is partially released into the medium during neutrophil stimulation by phorbol myristate acetate (PMA), presumably acting as one of the cytotoxic factors; and in its intracellular localization is presumably involved in the process of down regulation of native protein kinase C (PKC). Calpain, predominantly present in resting conditions in an inactive form, becomes activated in the course of neutrophil stimulation and appears to be involved both in the down regulation of native PKC as well as in the formation of a proteinase-activated kinase form, presumably derived from PKC and defined as PKC-M. Calpain once activated appears to be also involved in cytoskeleton rearrangement, through proteolytic degradation specifically oriented by substrate phosphorylation. Activation, down regulation of PKC, formation of the proteinase-activated kinase, as well as proteolytic processing of cytoskeleton have been demonstrated to be correlated to those biochemical responses which characterize neutrophil activation.

In recent years interest has increased concerning the characterization of the structural-functional properties and the identification of the physiological role of non-lysosomal intracellular proteinases. Among these, calpain, a calcium-dependent cysteine proteinase ubiquitously present in a variety of tissues and cells, has been most extensively investigated in terms of activation, regulatory mechanisms, specificity and biological function. This review discusses each of these points on the basis of the most recent results concerning the general characteristics of calpain activity, and its preferential site of action within the cell as related to the specific functions of the proteinase in different cell types. As with other proteinases, calpain has to be under a continuous spatial and temporal control, and the structural and functional properties of the natural calpain inhibitor, calpastatin, must also be considered. The calpain-calpastatin system is the functional proteolytic unit that governs the activity of this intracellular proteolytic system, which is tightly correlated to the control of calcium homeostasis and thereby to the biological process of transmembrane signalling.

The calmodulin antagonist N(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) has been examined as an inhibitor of superoxide anion production and granule exocytosis in phorbol ester (PMA)-activated neutrophils. Inhibition of the respiratory burst was observed at a concentration of W-7 identical to that required for inhibition of native protein kinase C (PKC), whereas the concentration required to inhibit the secretory response was found to correspond to that required for inhibition of the proteolytically converted fully active PKC. The IC50 of W-7 was in both cases 5 and 12 fold higher than that required for inhibition of calmodulin dependent kinases. The results confirm the essential role for the membrane-bound PKC in the production of O2- radicals and provide a clear evidence of the direct participation of the proteolytically activated cytosolic PKC to the secretory response of PMA activated neutrophils.

Human neutrophil calpain is a monomer of 85 kDa molecular weight. The proteinase shows an absolute requirement for Ca2+ with maximal catalytic activity at 0.1-0.2 mM Ca2+ and negligible activity at 1-5 microM Ca2+. At this concentration of Ca2+ neutrophil calpain becomes active and reaches 65% of its maximal catalytic activity following interaction with plasma membranes. The activation is fully reversible since the enzyme returns to its native, high Ca2+ requiring form following removal of the membranes. Membrane phospholipids appear to be the physiological compounds responsible for the promotion of such reversible activation. Unlike other Ca2+ dependent proteinases, neutrophil calpain does not undergo conversion to a low Ca2+ requiring form by limited autoproteolysis.

Publisher Summary This chapter discusses the properties and methods of assay of fructosediphosphatase (FDPase). It also discusses the purification of FDPase from different sources such as liver, kidney, muscles, and Candida utilis. The requirement of FDPase for gluconeogenesis has been firmly established by the observations with bacterial and human mutants referred to in an earlier section. However, the role of this enzyme in the regulation of carbohydrate metabolism remains to be clarified. Control of gluconeogenesis in liver appears to be exerted primarily a t the steps leading to the formation of phosphoenolpyruvate, but evidence has accumulated which suggests that control is also exerted at the level of fructose 1,6-diphosphate. Adenosine monophosphate (AMP), which is a specific inhibitor of FDPase in every organism examined except the slime mold, is also an activator of phosphofructokinase. High levels would therefore stimulate glycolysis, while low concentrations would favor gluconeogenesis. However, the levels of AMP have been found not to fluctuate significantly in fed or fasted animals, and other factors must therefore contribute to the metabolic control by this substance. One such factor may be the concentration of FDP, which itself inhibits FDPase, and which greatly enhances the inhibition by AMP. An additional regulatory mechanism is suggested by the low activity of purified FDPases in the neutral pH range and by the increases in neutral FDPase activity brought about by reagents, which modify sulfhydryl groups in the protein, particularly physiological agents such as CoA, acyl carrier protein, or homocystine.