Highlights of the 3rd International Conference on High Pressure Bioscience and Biotechnology.

The 3rd International Conference on High Pressure Bioscience and Biotechnology was held in the city of Rio de Janeiro from September 27 to September 30, 2004. The meeting, promoted by the International Association of High Pressure Bioscience and Biotechnology (IAHPBB), congregated top scientists and researchers from all over the world. In common, they shared the use of hydrostatic pressure for research, technical development, or industrial applications. The meeting consisted of invited lectures, contributed papers and a well-attended poster session. Very exciting discussions were held inside and outside the sessions, and the goals of discussing state-of-the-art data and establishing working collaborations and co-operations were fully attained.

Highlights of the 3rd International Conference on High Pressure Bioscience and Biotechnology

The 3rd International Conference on High Pressure Bioscience and Biotechnology was held in the city of Rio de Janeiro from September 27 to September 30, 2004. The meeting, promoted by the International Association of High Pressure Bioscience and Biotechnology (IAHPBB), congregated top scientists and researchers from all over the world. In common, they shared the use of hydrostatic pressure for research, technical development, or industrial applications. The meeting consisted of invited lectures, contributed papers and a well-attended poster session. Very exciting discussions were held inside and outside the sessions, and the goals of discussing state-of-the-art data and establishing working collaborations and co-operations were fully attained.

A novel role of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid as an activator of the phosphatase activity catalyzed by plasma membrane Ca2+-ATPase.

The hydrolysis of p-nitrophenyl phosphate catalyzed by the erythrocyte membrane Ca2+-ATPase is stimulated by low concentrations of the compound 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a classic inhibitor of anion transport. Enhancement of the phosphatase activity varies from 2- to 6-fold, depending on the Ca2+ and calmodulin concentrations used. Maximum stimulation of the pNPPase activity in ghosts is reached at 4-5 microM DIDS. Under the same conditions, but with ATP rather than pNPP as the substrate, the Ca2+-ATPase activity is strongly inhibited. Activation of pNPP hydrolysis by DIDS is equally effective for both ghosts and purified enzyme, and therefore is independent of its effect as an anion transport inhibitor. Binding of the activator does not change the Ca2+ dependence of the pNPPase activity. Stimulation is partially additive to the activation of the pNPPase activity elicited by calmodulin and appears to involve a strong affinity binding or covalent binding to sulfhydryl groups of the enzyme, since activation is reversed by addition of dithiothreitol but not by washing. The degree of activation of pNPP hydrolysis is greater at alkaline pH values. DIDS decreases the apparent affinity of the enzyme for pNPP whether in the presence of Ca2+ alone or Ca2+ and calmodulin or in the absence of Ca2+ (with 5 microM DIDS the observed Km shifts from 4.8 +/- 1.4 to 10.1 +/- 2.6, from 3.8 +/- 0.4 to 7.0 +/- 0.8, and from 9.3 +/- 0.7 to 15.5 +/- 1.1 mM, respectively). However, the pNPPase rate is always increased (as above, from 3.6 +/- 0.6 to 11.2 +/- 1.7, from 4.4 +/- 0.5 to 11.4 +/- 0.9, and from 2.6 +/- 0.6 to 18.6 +/- 3.9 nmol mg-1 min-1, in the presence of Ca2+ alone or Ca2+ and calmodulin or in the absence of Ca2+, respectively). ATP inhibits the pNPPase activity in the absence of Ca2+, both in the presence and in the absence of DIDS. Therefore, kinetic evidence indicates that DIDS does more than shift the enzyme to the E2 conformation. We propose that the transition from E2 to E1 is decreased and a new enzyme conformer, denoted E2*, is accumulated in the presence of DIDS.

ATPase and phosphatase activities are differentially inhibited by photo-oxidation of the sarcoplasmic reticulum Ca(2+)-ATPase.

We have already described that photo-oxidation of the sarcoplasmic reticulum Ca(2+)-ATPase with the halogenated dye erythrosin B produces inhibition of the ATPase activity (J.A. Mignaco et al., Biochemistry 35 (1996) 3886-3891). We now show that the Ca(2+)-dependent and Ca(2+)-independent p-nitrophenylphosphatase activities are also inhibited by this treatment. Modification of rapidly (< 10 min) oxidized residue(s) is responsible for the major loss of ATPase activity, whereas photo-inhibition of the phosphatase activities occurs more slowly (t1/2 20-30 min). Here we have focused on photo-inhibition of the Ca(2+)-independent pNPPase activity, and the counteracting effects of ATP and FITC. Following photo-oxidation, the Ca(2+)-independent pNPPase activity decreases monotonically. ATP partially protects against the inactivation of the pNPPase, whereas labeling the enzyme with FITC does not. However, the protective effect of ATP is completely abolished by the attached FITC. These data are interpreted in terms of two different sites that are susceptible to photo-oxidation and are involved in different events related to substrate hydrolysis.

Specificity and kinetic effects of nitrophenol analogues that activate myosin subfragment 1.

2,4-Dinitrophenol (DNP) activates the myosin ATPase of mammalian skeletal muscle in the presence of Ca2+ or Mg2+, and inhibits it when the bivalent cations are replaced by K+ and EDTA. Activation of Mg2+ATPase is abolished by the presence of unregulated actin. 3-Nitrophenol (3-NP) is also an activator, whereas other analogues (2-nitrophenol, 2-NP, and 4-nitrophenol, 4-NP) are much less effective. Concentrations required for their half-maximal effects (K0.5) range from 2 to 15 mM for 3-NP and DNP in the presence of different cations, and the sequence for the analogues is 3-NP<=DNP<<2-NP approximately 4-NP, which is apparently unrelated to either hydrophobicity or pK. DNP and 3-NP have almost identical effects on the ATPase activity of chymotryptic subfragment 1 as they do on myosin, which is an indication that their target is the globular head region rather than the tail, or the 18 kDa (regulatory) light chain. Analysis of the ATP concentration dependence for subfragment- 1 ATPase in the presence of Ca2+ or Mg2+ shows that DNP activates only at high substrate concentrations, becoming increasingly effective with ATP concentrations in the physiological range. At low substrate concentrations, DNP inhibits hydrolysis by increasing the apparent Km for ATP at the catalytic site. In the presence of Mg2+, it mimics the effect of actin, which increases the Km and accelerates the release of products following hydrolysis. At high substrate concentrations, activation by DNP appears to involve a kinetic component with low affinity for ATP that can increase the overall reaction rate by a factor of 2- to 9-fold, depending on the bivalent cation. This low-affinity component is either induced by the drug (in the presence of Mg2+) or shifted by the drug to a lower ATP concentration range (in the presence of Ca2+).

Effects of photo-oxidizing analogs of fluorescein on the sarcoplasmic reticulum Ca2+-ATPase. Functional consequences for substrate hydrolysis and effects on the partial reactions of the hydrolytic cycle.

Erythrosin B was used to photo-oxidize the sarcoplasmic reticulum Ca2+-ATPase. The ATPase activity is rapidly and irreversibly inhibited by photo-oxidation with erythrosin. This inhibition is protected by the presence of ATP during the photo-oxidation period. After photo-oxidation, the steady-state phosphorylation by ATP remains almost unchanged, whereas phosphorylation by inorganic phosphate is impaired. The pseudo-first order rate constants for phosphorylation by 15 microM ATP at 25 degrees C are strongly inhibited when starting from either a Ca2+-bound or a Ca2+-free enzyme form, decreasing from 145 to 23 s-1 for the Ca2+-bound form and from 50 to 18 s-1 for the Ca2+-free form. Concurrently, the rate constants for dephosphorylation are also severely inhibited, changing from a fast double exponential to a very slow single exponential decay in the reverse direction and from a moderately slow single to a very slow single exponential decay in the forward direction. Ca2+ binding data show that the phosphorylated intermediate formed by the photo-oxidized enzyme contains two occluded Ca2+, and TNP-ATP fluorescence measurements indicate that it accumulates in a E1-P.Ca2-like conformation. Protection by ADP against glutaraldehyde-induced cross-linking indicates that ADP binding to Ca2+-ATPase is not impaired by photo-oxidation nor by free erythrosin. These data support the view that an ADP-insensitive, Ca2+-bound, slowly interconverting phosphoenzyme is formed. Thus, photo-oxidation with erythrosin B leads to impairment of phosphoryl transfer reactions and related conformational changes.

Two simultaneous binding sites for nucleotide analogs are kinetically distinguishable on the sarcoplasmic reticulum Ca(2+)-ATPase.

Erythrosin B and eosin Y stimulate p-nitrophenyl phosphate hydrolysis by purified sarcoplasmic reticulum Ca(2+)-ATPase by nearly 2-3 fold in the presence of Ca(2+). This stimulation is not due to the change on the apparent affinity for substrate but is indeed due to acceleration of the turnover rate of the enzyme. Stimulation reaches a maximum at approximately 5 microM erythrosin or 20 microM eosin and is strictly dependent on the presence of Ca(2+) in reaction media, while higher concentrations of dye progressively inhibit phosphatase activity. Labeling with fluorescein isothiocyanate (FITC) largely shifts the Km for p-nitrophenyl phosphate (pNPP) and completely abolishes the stimulation of phosphatase activity induced by erythrosin in the presence of Ca(2+), apparently by FITC impairing dye binding to an activator site and allowing only manifestation of an inhibitory binding site. In the absence of Ca(2+), both erythrosin and eosin inhibit pNPP hyrolysis with Ic50 values 3-4 fold higher than the maximally stimulatory enzyme with FITC, which by its turn does not affect pNPPase activity in absence of Ca(2+). It is suggested that stimulation and inhibition of phosphatase activity are related to two simultaneous and physically different nucleotide analog binding sites.