An examination of the factors involved in determining phosphatase activities in estuarine water. 1: Analytical procedures

The effects of assay pH, magnesium addition, temperature, sodium azide, and of sample storage and filtration on the apparent phosphatase activities in natural water bodies, and in particular estuaries, have been examined. Phosphatase activity was assessed by monitoring the cleavage of p-nitrophenol from p-nitrophenyl phosphate (pNPP). Magnesium concentrations in natural waters were found to be generally in excess of the requirement of the assay procedure. Several pH maxima occurred for phosphatase activities in the estuarine waters examined. These optima were not constant with respect to sampling stations or sampling time. This was considered to be, in part, the result of differences in the biomass composition. Sample storage times and temperatures employed in the present study did not influence the phosphatase activities of these low biomass waters. Phosphatase activities increased with increased incubation temperature. The rate of orthophosphate release from pNPP during incubation was linear over time to 120 h. Sodium azide was found to be an adequate preservative when long incubation times were required. The effects of assay conditions are discussed in relation to the assay procedures and data interpretation.     

Properties and Regional Distribution of Pyruvate Dehydrogenase Kinase in Rat Brain

A method is described to measure directly in rat brain the activity of pyruvate dehydrogenase kinase (PDHa kinase; EC 2.7.1.99), which catalyzes the inactivation of pyruvate dehydrogenase complex (PDHC, EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3). The activity showed the expected dependence on added ATP and divalent cation, and the expected inhibition by dichloroacetate, pyruvate, and thiamin pyrophosphate. These results, and the properties of pyruvate dehydrogenase phosphate phosphatase (EC 3.1.3.43), indicate that the mechanisms of control of phosphorylation of PDHC seem qualitatively similar in brain to those in other tissues. Regionally, PDHa kinase is more active in cerebral cortex and hippocampus, and less active in hypothalamus, pons and medulla, and olfactory bulbs. Indeed, the PDHa kinase activity in olfactory bulbs is uniquely low, and is more sensitive to inhibition by pyruvate and dichloroacetate than that in the cerebral cortex. Thus, there are significant quantitative differences in the enzymatic apparatus for controlling PDHC activity in different parts of the brain.     

Hormone-responsive cells derived from human dental papilla: Characterization in vitro and in vivo in diffusion chambers

Summary Cells of the dental papilla are capable of odontoblastic, fibroblastic, and endothelial differentiation and formation of dentin and the dental pulp. In the present study dental papilla cells, obtained from human tooth buds (HDP cells), were cultured in vitro through 3 to 7 passages. After exposure to prostaglandin E₂ there was a marked decrease in intracellular cyclic AMP (cAMP) levels as compared to hormone-free controls. Parathyroid hormone and calcitonin had stimulatory effects with 1 and 2 log increases in cAMP, respectively. The HDP cells showed moderate activity of alkaline phosphatase, 1 log higher than that of hamster kidney fibroblasts (BHK 13) and 1 log lower than that of osteoblastic osteosarcoma cells (ROS 17/2). When cultured for 4 or 8 wk in diffusion chambers (DC) implanted in athymic mice, many of the HDP cells underwent odontoblastic morphodifferentiation with very long, single processes extending into the matrix. This matrix contained banded and unbanded collagen fibers. Neither light nor electron microscopy of the DC content revealed mineral deposits. These results suggest that HDP cells have an intrinsic potential for partial odontoblastic differentiation; inductive signals like those originating from odontogenic epithelium are probably essential for the completion of hard tissue formation.     

Dephosphorylation and reactivation of phosphorylated purified ox-kidney branched-chain dehydrogenase complex by co-purified phosphatase

The branched-chain 2 oxoacid dehydrogenase complex has been purified from well-washed ox-kidney mitochondria together with branched-chain dehydrogenase kinase. The complex was inactivated and phosphorylated by ATP in about 5 min at 30 degrees C. After hydrolysis of ATP by a contaminating ATPase (5-10 min) the complex was dephosphorylated and reactivated. Dephosphorylation and reactivation were linearly correlated. Reactivation was dependent upon Mg2+ (K0.5 greater than 1 mM) and inhibited completely by 50 mM fluoride. Reactivation and dephosphorylation are attributed to a mitochondrial branched-chain dehydrogenase phosphatase.     

Effect of metabolic acidosis on phosphate transport by the renal brush-border membrane

Metabolic acidosis produces a phosphaturia which is independent of parathyroid hormone or dietary phosphorus intake. To study the underlying mechanism, inorganic phosphate (P_i) and glucose transport were studied in brush-border membrane vesicles prepared from the renal cortex of parathyroidectomized rats gavaged for three days with either 7.5 ml of 1.6% NaCl (control) or 1.5% NH₄Cl (acidosis). At killing, blood pH and plasma bicarbonate were $\text{7.36 ± 0.01}$ and $\text{21.8 ± 0.8}\text{mequiv./l}$, respectively, in control and $\text{7.12 ± 0.03}$ ($\text{P < 0.01}$) and $\text{11.1 ± 1.2}$ ($\text{P < 0.01}$) in acidotic rats. Serum P_i was similar in both groups, while 24 h urine P_i excretion was higher in the acidotic group ($\text{P < 0.01}$). Peak sodium-dependent uptake of P_i, measured after 1.5 min of incubation, was higher in controls than acidotic rats ($\text{4442 ± 464}$ vs. $\text{2412 ± 259}\text{pmol/mg}$ protein, $\text{P < 0.01}$), whereas peak glucose uptake at 1.5 min was not significantly different between the groups. Equilibrium values for P_i and glucose uptake were similar in the two groups. $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for P_i uptake in the control and acidotic animals were not different, 0.036 and 0.040 mM, respectively. By contrast, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ was higher in controls than in the acidotic group, 3.13 vs. 1.15 nmol/mg protein per 15 s. These results suggest that metabolic acidosis directly inhibits P_i uptake by the brush border of the proximal tubule by decreasing the availability of P_i carriers of the renal brush-border membrane.     

Acid phosphatase from maize scutellum: Negative cooperativity suppression by glucose

Acid phosphatase purified from maize scutellum, upon acylation with succinic anhydride, still shows negative co-operativity for the hydrolysis of glucose-6-phosphate at pH 5.4. This phenomenon is abolished by glucose, for both native and succinylated enzymes, through stimulation of the initial velocities at sub-optimal substrate concentrations. However, negative co-operativity for the enzymatic hydrolysis of p-nitrophenylphosphate at pH 5.4 is suppressed only at high concentrations of glucose. Furthermore, the hydrolysis of p-nitrophenylphosphate is noncompetitively inhibited (low affinity form of the enzyme molecule) by glucose, which suggests the existence of different substrate binding sites.     

Excrétion de phosphatase acide par les cellules mucipares de la branchie de Mytilus edulis L.

Résumé Dans toutes les cellules mucipares de la branchie de Mytilus edulis L., quelques grains de secretion montrent une activité phosphatasique acide, décelée par la méthode cytochimique de Gomori appliquée au microscope électronique. A la proximité de ces grains se trouvent des dictyosomes golgiens où une activité phosphatasique se décèle dans les parties latérales des cisternes et dans les vésicules qui en émanent. Au moment de l'excrétion, tous les grains de sécrétion confluent et la phosphatase acide active est incluse dans l'amas de mucus expulsé dans la cavité palléale. Le rôle digestif de ces amas — et non purement mécanique — peut ainsi être considéré comme hautement probable. De telles manifestations enzymatiques sont totalement absentes dans le tapis muqueux ainsi que dans les cellules qui le constituent.

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Excretion of acidic phosphatase by the goblet-cells of the gill of Mytilus edulis L.An electron microscopic study

Summary Acidic phosphatase activity detected by the Gomori method applied to E.M., has been found in a few secretion granules of each goblet cell of the gill of Mytilus edulis L. In the close proximity of those granules golgian dictyosomes always occur, with acidic phosphatase activity in the lateral parts of the cisternae and in the vesicles arising from the latter. When the mucus is extruded, active and non active secretion granules are mixed and thus, acidic phosphatase activity is regularly found in the mucous tufts extruded into the palleal cavity. Therefore the digestive role — and not merely mechanic — of the mucous tufts may be considered as highly probable. Conversely, the secretion granules from which arises the continuous mucous cover of the epithelium and this cover also, are entirely devoid of any acidic phosphatase activity.

     

Effects of seven different mutations in the pho1 gene on enzymatic activity, glycosylation and secretion of acid phosphatase in Schizosaccharomyces pombe

Summary Structural gene mutants of the cell-surface glycoprotein acid phosphatase of Schizosaccharomyces pombe were analysed to define structural determinants that are responsible for enzymatic activity, N-glycosylation and secretion. All seven defined mutations cause a single amino acid substitution in the mature acid phosphatase protein and destroy the enzymatic activity. The mutational lesions are distributed throughout the pho1 gene. A ser to phe substitution at position 349 abolishes enzymatic activity only and does not affect glycosylation and secretion. Two mutations create a new N-glycosylation site by substitution of pro at position 56 by phe and ser, respectively. This new site is apparently used in the mutants. Their core-glycosylated acid phosphatase is slightly larger than that of the wild type. Overglycosylation seems not to affect secretion. Four different mutations (a gly to asp substitution at position 281 and ser to phe substitutions at positions 150, 271 and 277) cause intracellular accumulation of enzymatically inactive core-glycosylated acid phosphatase precursor. These mutational lesions apparently block transport of acid phosphatase from the endoplasmic reticulum to the Golgi apparatus.     

Change in the cellular localization of alkaline phosphatase by alteration of its carboxy-terminal sequence

Summary Alkaline phosphatase (AP) is secreted into the medium when the carboxy-terminal 25 amino acids are replaced by the 60 amino acid carboxy-terminal signal peptide (HlyA_s) ofEscherichia coli haemolysin (HlyA). Secretion of the AP-HlyA_s fusion protein is dependent on HlyB and HlyD but independent of SecA and SecY. The efficiency of secretion by HlyB/HlyD is decreased when AP carries its own N-terminal signal peptide. Translocation of this fusion protein into the periplasm is not observed even in the absence of HlyB/HlyD. The failure of the Sec export machinery to transport the latter protein into the periplasm seems to be due in part to the loss of the carboxy-terminal sequence of AP since even AP derivatives which do not carry the HlyA signal peptide but lack the 25 C-terminal amino acids of AP are localized in the membrane but not translocated into the periplasm.     

Molecular characterization of the lysosomal acid phosphatase fromDrosophila melanogaster

InDrosophila, unlike humans, the lysosomal acid phosphatase (Acph-1) is a non-essential enzyme. It is also one of the most rapidly evolving gene-enzyme systems in the genus. In order to determine which parts of the enzyme are conserved and which parts are apparently under little functional constraint, we cloned the gene fromDrosophila melanogaster via a chromosomal walk. Fragments from the gene were used to recover an apparently full-length cDNA. The cDNA was subcloned into aDrosophila transformation vector where it was under the control of the 5 promoter sequence of thehsp-70 gene. Three independent transformants were obtained; in each, Acph-1 expression from the cDNA was constitutive and not dependent on heat shock, as determined by densitometric analyses of the allozymic forms of the enzyme. The pattern of expression indicates thehsp-70 and endogenousAcph-1 promoters act together in some, but not all, tissues. The sequence of the cDNA was determined using deletions made with exonuclease III, and primers deduced from the cDNA sequence were used to sequence the genomic clone. Five introns were found, and putative 5 up-stream regulatory sequences were identified. Amino acid sequence comparisons have revealed several highly conserved motifs betweenDrosophila Acph-1 and vertebrate lysosomal and prostatic acid phosphatases.