Role of a nonmitochondrial Ca2+ pool in the synergistic stimulation by cyclic AMP and vasopressin of Ca2+ uptake in isolated rat hepatocytes.

The subcellular distribution of 45Ca2+ accumulated by isolated rat hepatocytes exposed to dibutyryl cyclic AMP (dbcAMP) followed by vasopressin (Vp) was studied by means of a nondisruptive technique. When treated with dbcAMP followed by vasopressin, hepatocytes obtained from fed rats accumulated an amount of Ca2+ approximately fivefold higher than that attained under control conditions. Ca2+ released from the mitochondrial compartment by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) accounted for only a minor portion of the accumulated Ca2+. The largest portion was released by the Ca2+ ionophore A23187 and was attributable to a nonmitochondrial compartment. DbcAMP + Vp-treatment also caused a maximal stimulation of glucose production and a twofold increase in cellular glucose 6-phosphate levels. In hepatocytes obtained from fasted rats, dbcAMP + Vp-stimulated Ca2+ accumulation was lower, although with the same subcellular distribution, and was associated with a minimal glucose production. In the presence of gluconeogenetic substrates (lactate plus pyruvate) hepatocytes from fasted rats were comparable to cells isolated from fed animals. However, Ca2+ accumulation and glucose 6-phosphate production could be dissociated in the absence of dbcAMP, in the presence of lactate/pyruvate alone. Under this condition in fact Vp induced only a minimal accumulation of Ca2+ in hepatocytes isolated from fasted rats, although glucose production was markedly increased. Moreover, treatment of fed rat hepatocytes with 1 mM ATP caused a maximal activation of glycogenolysis, but only a moderate stimulation of cellular Ca2+ accumulation. In this case, sequestration of Ca2+ occurred mainly in the mitochondrial compartment. By contrast, the addition of ATP to dbcAMP-pretreated hepatocytes induced a large accumulation of Ca2+ in a nonmitochondrial pool. Additional experiments using the fluorescent Ca2+ indicator Fura-2 showed that dbcAMP pretreatment can enlarge and prolong the elevation of cytosolic free Ca2+ caused by Vp. A nonmitochondrial Ca2+ pool thus appears mainly responsible for the Ca2+ accumulation stimulated by dbcAMP and Vp in isolated hepatocytes, and cyclic AMP seems able to activate Ca2+ uptake in such a nonmitochondrial pool.     

Analysis of the activation process of porcine procarboxypeptidase B and determination of the sequence of its activation segment

The molecular events which lead to the proteolytic transformation of porcine procarboxypeptidase B (PCPB) in carboxypeptidase B (CPB) have been determined. Among pancreatic and other tested proteinases, trypsin is the only one capable of generating carboxypeptidase B activity from the zymogen, in vitro. In the first step of this process, trypsin produces cleavage at the boundary between the activation region and the CPB region. Subsequently, a definite sequence of cleavages occurs at the C-terminal end of the released activation segment of 95 residues, giving rise to characteristic intermediates and to a proteolytically resistant activation fragment of 81 residues. In this process, the newly formed CPB participates in the quick-trimming of the released activation peptides. Only a single CPB species is formed in the activation process. This fact and the inability of the released activation peptides to inhibit CPB--and, therefore, their inability to slow down the kinetics of appearance of CPB activity--are two important characteristics differentiating between the activation processes of procarboxypeptidases A and B. The sequence of the 95 residues (MW = 12,835) of the activation region of porcine PCPB has also been deduced, largely from the information obtained by Edman degradation of its fragments and in part by considerations of homology with the rat precursor. The porcine PCPB activation region contains a high percentage of acidic residues, lacks cysteines, methionines, and side-chain posttranslational modifications, and presents a low but significant homology (31%) with the corresponding sequence of porcine procarboxypeptidase A.     

The most conserved nuclear-encoded polypeptide of cytochrome c oxidase is the putative zinc-binding subunit: primary structure of subunit V from the slime mold Dictyostelium discoideum.

A full-length 515 base pairs cDNA for cytochrome c oxidase subunit V of D. discoideum was isolated from a lambda gt11 expression library. The encoded polypeptide, whose identity was confirmed by partial protein sequencing, is 119 amino acids long (Mr = 13,352) and does not contain a cleavable presequence. The protein, which is homologous to human subunit Vb and yeast subunit IV, exhibits the highest degree of sequence conservation found among nuclear-encoded subunits of cytochrome c oxidase from distantly related organisms. All the invariant residues are clustered in two regions of the C-terminus which include the putative amino acids involved in the coordination of the Zn ion tightly associated to eukaryotic oxidase.     

Assay of β-galactosidase activity by Flow Injection Analysis (FIA)

Summary A novel method to analyze -galactosidase by Flow Injection Analysis is presented with a linear working range extended to at least 2150 U/mL, being the detection limit 25 U/mL with 55 samples per hour frecuency and a BSD of 0.954% versus 2.4% obtained by manual assay. The method was tested with optimal results with samples from Escherichia coli cultures producing -galactosidase.     

Comparison of the hydrological characteristics of three small experimental holm oak forested catchments in NE Spain in relation to larger areas

Precipitation and streamflow have been measured in three small (0.04–0.52 km²) experimental catchments covered by dense holm oak (Quercus ilex L.) forests. Two of them are in the Prades mountains and one in the Montseny mountains (NE Spain). Here we test the hydrological representativeness of these catchments against the streamflow record at two nearby larger (34–60 km²) catchments, one from each massif. Comparisons of (i) annual streamflow, (ii) seasonal distribution of streamflow, and (iii) flow duration curves were made. At Prades, for the period of common record, mean annual precipitation was about 580 mm, and mean annual streamflow 44–81 mm at the two experimental catchments and 102 mm at the larger one. Most streamflow occurred during winter and spring in the three catchments. At Montseny, rainfall was higher, and mean annual streamflow was 495 mm in the experimental catchment, and 760 mm in the larger catchment, though these data were obtained in different periods in each catchment. Streamflow was roughly equal in autumn, winter and spring. At both sites flow duration curves were fairly similar in the small experimental catchments and the larger catchments. The higher streamflow at Montseny is reflected in its flow duration curves being well above those at Prades. The experimental catchments at Prades are thus fairly representative of the larger nearby catchment for the investigated hydrological characteristics. At Montseny, hydrological differences between the experimental catchment and the larger catchment are probably due to the higher mean altitude of the latter and to the non-overlapping periods of their streamflow records.     

Changes in the protein synthesis pattern during a nutritional shift-down transition in Saccharomyces cerevisiae

In Saccharomyces cerevisiae cells (strain A364A) during a shift-down from glucose to raffinose, a rapid reduction in the rate of RNA accumulation was observed whereas the rate of protein accumulation was unaffected for at least 2 h. Following the transition the percentage of unbudded cells slightly increased and the cell volume distribution showed a newly formed subpopulation of smaller cells. To study the effects of the shift-down on the protein synthesis pattern, total [35S]-methionine pulse-labeled extracts were fractionated by high-resolution two-dimensional gel electrophoresis. The synthesis of two classes of proteins (I and II) was modulated during the transitory state of growth: one positively, the other negatively. Two polypeptides of 57 kDa showed the most dramatic increase in synthesis during the shift-down. Also a heat-shock protein (HSP 256) appeared to be positively correlated to the shift-down transition.     

A tetrameric iron superoxide dismutase from the eucaryote Tetrahymena pyriformis

An iron-containing superoxide dismutase has been purified from the protozoan Tetrahymena pyriformis. It has a molecular weight of 85,000 and is composed of four subunits of equal size. The tetramer contains 2.5 g atoms of ferric iron. Visible absorption and electron spin resonance spectra closely resemble those of other iron-containing superoxide dismutases. The amino acid sequence of the iron superoxide dismutase was determined. Each subunit is made up of 196 residues, corresponding to a molecular weight of 22,711. Comparison of the primary structure with the known sequences of other iron-containing superoxide dismutases reveals a relatively low degree of identity (33-34%). However, a higher percentage identity is found with mammalian manganese-containing superoxide dismutases (41-42%). The amino acid sequence is discussed in consideration of residues that may distinguish iron from manganese or dimeric from tetrameric superoxide dismutases.     

Proteins required for the binding of mitrochondrial ATPase to the mitochondrial inner membrane.

1. Isolated F1 (mitochondrial ATPase) binds to urea-treated submitochondrial particles suspended in sucrose/Tris/EDTA with a dissociation constant of 0.1 muM. 2. About one-third of the F1 and the oligomycin-sensitivity conferring protein (OSCP) are lost during preparation of submitochondrial particles prepared at high pH (A particles). None is lost from particles treated with trypsin (T particles). 3. After further treatment with alkali of urea-treated particles, binding of F1 requires the addition of OSCP. Maximum binding is reached when both OSCP and Fc2 are added. The concentration of F1-binding sites in the presence of both OSCP and Fc2 is about the same as that in TU particles. 4. After further extraction with silicotungstate of urea- and alkali-treated particles, OSCP no longer induces binding of F1, unless Fc2 is also present. Fc2 induces binding in the absence of OSCP but with a lower binding constant and, in contrast to results under all the other conditions studied in this paper, the ATPase activity is oligomycin insensitive. 5. It is tentatively concluded that OSCP is the binding site for F1 and Fc2 is the binding site for OSCP.