Electron-cytochemical localization of alkaline phosphatase to G cells of Necturus maculosus antrum

Summary Electron-cytochemical localization of alkaline phosphatase activity was performed on G cells of Necturus maculosus antral mucosa. Alkaline phosphatase activity was localized to the nuclear membrane, the Golgi/endoplasmic reticulum, and the limiting membranes of G cell peptide-secretion vesicles. There was no specific localization of alkaline phosphatase activity to the plasma membrane. Treatment of the tissues with levamisole (an alkaline phosphatase inhibitor) did not markedly reduce the specific alkaline phosphatase activity. Specific lead deposition was reduced by removal of the substrate from the reaction mixture. The results from this study on N. maculosus G cells demonstrate that alkaline phosphatase activity can be found in a non-mammalian gastric endocrine cell and that specific activity was localized primarily to those intracellular structures involved with protein biosynthesis.     

Protein phosphatase activities in vivo in Xenopus laevis oocyte: inhibition by okadaic acid

Protein phosphatase activities were analyzed in vivo in Xenopus oocytes. The dephosphorylation of microinjected beta casein was inhibited when the tumor promoter okadaic acid was microinjected into oocytes. Inhibition was dose dependent and reversible; 50% of activity was recovered 15-30 minutes after microinjection.     

The role of phospholamban in the regulation of calcium transport by cardiac sarcoplasmic reticulum

The calcium transport mechanism of cardiac sarcoplasmic reticulum (SR) is regulated by a phosphoregulatory mechanism involving the phosphorylation-dephosphorylation of an integral membrane component, termed phospholamban. Phospholamban, a 27,000 Da proteolipid, contains phosphorylation sites for three independent protein kinases: 1) cAMP-dependent, 2) Ca²⁺-calmodulin-dependent, and 3) Ca²⁺-phospholipid-dependent. Phosphorylation of phospholamban by any one of these kinases is associated with stimulation of the calcium transport rates in isolated SR vesicles. Dephosphorylation of phosphorylated phospholamban results in the reversal of the stimulatory effects produced by the protein kinases. Studies conducted on perfused hearts have shown that during exposure to beta-adrenergic agents, a good correlation exists between the in situ phosphorylation of phospholamban and the relaxation of the left ventricle. Phosphorylation of phospholamban in situ is also associated with stimulation of calcium transport rates by cardiac SR, similar to in vitro findings. Removal of beta-adrenergic agents results in the reversal of the inotropic response and this is associated with dephosphorylation of phospholamban. These findings indicate that a phospho-regulatory mechanism involving phospholamban may provide at least one of the controls for regulation of the contractile properties of the myocardium.     

Divalent cation effects on calcineurin phosphatase: differential involvement of hydrophobic and metal binding domains in the regulation of the enzyme activity

Summary The effects of divalent metals, metal chelators (EDTA, EGTA) and sodium dodecyl sulfate were investigated on the phosphatase activity of isolated bovine brain calcineurin assayed in the absence (called intrinsic) and presence of calmodulin. Intrinsic phosphatase was increased by Mn²⁺, was unaffected by Mg²⁺, Ca^2–, and Ba⁺, and was markedly inhibited by Ni^2–, Fe²⁺, Zn²⁺ and Cu^2–. When assayed in the presence of calmodulin, many divalent metals (Ni^2–, Zn²⁺, Pb²⁺, Cd²⁺), besides Mn²⁺, increased modestly the phosphatase activity at low concentrations (10–100 M) and inhibited it markedly at high concentrations. Ca^2–-calmodulin stimulated phosphatase activity was antagonized by Ni²⁺, Zn²⁺, Fe²⁺, Cu²⁺, Pb²⁺, at low concentrations (50 M), and by Ba²⁺, Cd²⁺ at slightly higher concentrations (> 100 M); Mn²⁺ and Co^2– (50 M to 1 mM) in fact augmented it. EDTA and EGTA in a concentration and time dependent fashion inhibited the intrinsic phosphatase activity, particularly that of trypsinized calcineurin. SDS in low concentrations (0.005%) augmented the phosphatase activity and inhibited it at high concentrations. Mn²⁺ (± calmodulin) and Ca²⁺ only with calmodulin present increased the phosphatase activity assayed with low concentrations of SDS. The EDTA dependent inhibition of intrinsic phosphatase was almost abolished in assays containing SDS. Prior exposure of calcineurin to Mn²⁺ led to a high activity conformation state of calcineurin that was long-lived or pseudo-irreversible. Such Mn²⁺-activated state of calcineurin exhibited no discerbible change in the affinity towards myelin basic protein or its inhibition by trifluoperazine. At alkaline pH, Mg²⁺ supported the intrinsic phosphatase activity, although to a lesser degree than Mn²⁺. The latter cation, compared to Mg²⁺ and Ni²⁺, was also a more powerful stimulator of the calcineurin phosphatase assayed with histone (III-S) and myosin light chain as substrates.     

Heat-stable alkaline phosphatase as a marker for human and monkey type-I pneumocytes

Summary The expression of the heat-stable isoenzyme of alkaline phosphatase in the human and monkey (Macaca mulatta, M. fascicularis) lung was investigated at the light- and electron-microscopic level, using cytochemical techniques and immunocytochemical procedures based on monoclonal and polyclonal antibodies against human term-placental alkaline phosphatase. Both in man and monkey, the enzyme was present in type-I pneumocytes. In the monkey, the enzyme was found in all type-I cells. In man, strong staining was observed only in some type-I cells and in certain cuboidal respiratory bronchiolar cells. Staining was localized on the apical and basal plasma membrane, in apical and basal caveolae, and in the underlying basement membrane. The level of heat-stable alkaline phosphatase expression in the human lung was 10-fold lower than in the monkeys studied. In human fetal lung, the onset of heat-stable alkaline phosphatase expression was associated with the development of the alveolar epithelium from 17–20 weeks gestation onward. It is concluded that: (1) heat-stable alkaline phosphatase is a specific constitutent of type-I pneumocytes in man and monkeys; and (2) its subcellular localization may explain its rapid appearance in the circulation under certain conditions.This work was supported by grants from the Fonds voor Kankeronderzoek van de Algemene Spaar- en Lijfrentekas, Nationale Loterij-FGWO (Grant No. 9.0005.84), the National Program for Reinforcement of the Scientific Research (PREST/UIA 04) and a research grant from the University of Antwerp     

白皮松茎次生韧皮部中蛋白细胞的细胞化学研究

蛋白细胞是裸子植物次生韧皮部中唯一与筛胞发生单侧筛域联系的一类特殊的薄壁组织细胞,并且具有相当高的代谢潜势。本文在白皮松次生韧皮部蛋白细胞解剖学和细胞学研究的基础上进行了蛋白细胞中酸性磷酸酯酶和 ATP 酶的定位,首次报道成熟蛋白细胞中存在相当高的 ATP 酶活性的研究结果。     

Solubility properties of alkaline phosphatase from matrix vesicles

Alkaline phosphatase has been extracted from matrix vesicles of a calcifying cartilage with 0.15 M KCl, 0.4 M guanidinium chloride and 0.05 M deoxycholate/50% butanol mixture. The catalytic properties of the three extracts have been compared. Although the highest amount of enzyme activity is extracted with the latter reagent (55%), some of it is also extracted with KCl (11%) and guanidinium (7%). By submitting isolated matrix vesicles to a short time sonication the distribution pattern of the alkaline phosphatase activity in the extracts is clearly modified, as the amount extracted with KCl increases from 14 to 50% and the portion extracted with deoxycholate decreases from 55 to 27% of the total enzyme activity of matrix vesicles. The enzymatic preparations were comparable on the basis of specific activities, affinity for the substrates (p-nitrophenylphosphate, ATP), thermostability, sensitivity to inhibitors and activators. By electrofocusing a value of pI = 4.15 was found for the alkaline phosphatase of matrix vesicles independently of the extraction medium. These results contradict the concept that alkaline phosphatase is exclusively an intrinsic membrane protein.     

Protein Uptake and Digestion in Bloodstream and Culture Forms of Trypanosoma brucei*

SYNOPSIS. The mechanisms of ferritin uptake and digestion differ in bloodstream and culture forms of Trypanosoma brucei. Ferritin enters bloodstream forms from the flagellar pocket by pinocytosis in large spiny-coated vesicles. These vesicles become continuous with straight tubular extensions of a complex, mostly tubular, collecting membrane system where ferritin is concentrated. From the collecting membrane system the tracer enters large digestive vacuoles. Small spiny-coated vesicles, which never contain ferritin, are found in the Golgi region, fusing with the collecting membrane system, and around the flagellar pocket. Acid phosphatase activity is present in some small spiny-coated vesicles which may represent primary lysosomes. This enzymic activity is also found in the flagellar pocket, pinocytotic vesicles, the collecting membrane system, the Golgi (mature face), and digestive vacuoles of bloodstream forms. About 50% of the acid phosphatase activity of blood forms is latent. The remaining nonlatent activity is firmly cell-associated and probably represents activity in the flagellar pocket. The structures involved in ferritin uptake and digestion are larger and more active in the short stumpy than in the long slender bloodstream forms. The short stumpy forms also have more autophagic vacuoles. No pinocytotic large, spiny-coated vesicles or Golgi-derived, small spiny-coated vesicles are seen in culture forms. Ferritin leaves the flagellar pocket of these forms and enters small smooth cisternae located just beneath bulges in the pocket membrane. The tracer then passes through a cisternal collecting membrane network, where it is concentrated, and then into multivesicular bodies. In the culture forms, acid phosphatase activity is localized in the cisternal system, multivesicular bodies, the Golgi (mature face), and small vesicles in the Golgi and cisternal regions. The flagellar pocket has no acid phosphatase activity, and almost all the activity is latent in these forms. The culture forms do not release acid phosphatase into culture medium during 4 days growth. Uptake of ferritin by all forms is almost completely inhibited by low temperature. These differences among the long slender and short stumpy bloodstream forms and culture forms are undoubtedly adaptive and reflect different needs of the parasite in different life cycle stages.     

Evaluation of serum parameters relevant to vitamin D status in tamarins

Serum levels of 25(OH)D, alkaline phosphatase (AP), and parathormone (PTH) were evaluated to investigate the vitamin D requirement of Saguinus fuscicollis. Diets with various vitamin D content were fed 4 weeks and longer. The values of 25(OH)D (30-300 nmol/l), AP (less than 300 U/l), and PTH (less than or equal to 1,000 equl/l) considered as normal were obtained with 2,000 IU D3/kg diet, or 33 IU/animal/day, which we regard as the level required. Animals depleted of vitamin D for 215 days developed a secondary hyperparathyroidism.     

Inactivation and Reactivation of the Multifunctional Calmodulin-Dependent Protein Kinase from Brain by Autophosphorylation and Dephosphorylation: Involvement of Protein Phosphatases from Brain

The multifunctional calmodulin-dependent protein kinase (calmodulin-kinase) from rat brain was autophosphorylated in a Ca2+- and calmodulin-dependent manner. The activity of the autophosphorylated enzyme was independent of Ca2+ and calmodulin. Calmodulin-kinase was dephosphorylated by protein phosphatase C from bovine brain, which is the catalytic subunits of protein phosphatases 1 and 2A. The holoenzyme of protein phosphatase 2A was also involved in the dephosphorylation of the enzyme. The autophosphorylated sites of calmodulin-kinase were universally dephosphorylated by protein phosphatase C. Calmodulin-kinase was inactivated and reactivated by autophosphorylation and dephosphorylation, respectively. Furthermore, the regulation of calmodulin-kinase by autophosphorylation and dephosphorylation was observed using calmodulin-kinase from canine heart. These results suggest that the activity of calmodulin-kinase is regulated by autophosphorylation and dephosphorylation, and that the regulation is the universal phenomenon for many other calmodulin-kinases in various tissues.