A new synthesis of benzoyl phosphate: A substrate for acyl phosphatase assay

Summary A new method for the synthesis of benzoyl phosphate was reported. The advantages are: 1. more rapid procedure; 2. lower cost; 3. higher yield.This work was supported by grants from the Italian Consiglio Nazionale delle Ricerche and from the Ministero della Pubblica Istruzione.     

2-Methoxybenzoyl phosphate: a new substrate for continuous fluorimetric and spectrophotometric acyl phosphatase assays

A new aromatic acyl phosphate, 2-methoxybenzoyl phosphate, has been synthesized. The compound shows an intrinsic fluorescence; it displays an intense emission band at 390 nm upon excitation in the near UV region. This band practically disappears after hydrolysis of the product. On the other hand, the product displays differences in the near UV absorption spectra measured before and after hydrolysis. The at 301 nm is 2720 M^–1 cm^–1, a value that is 4.3-fold higher than that of benzoyl phosphate (the usual substrate for acylphosphatase assay) at 283 nm. The main kinetic parameters of three different acylphosphatase molecular forms (the muscular isoenzyme and two subtypes of the organ common isoenzyme) were determined using both benzoyl phosphate and 2-methoxybenzoyl phosphate as substrates, and then compared. These kinetic data and the UV absorption and fluorescence properties of 2-methoxybenzoyl phosphate sugest that this compound has better substrate features than benzoyl phosphate, and can be used for both high sensitivity continuous fluorimetric and UV absorption spectrophotometric assays of acylphosphatase.     

Alkaline phosphatase activity in normal and denervated skeletal muscle

Summary Changes in the specific activity of alkaline phosphatase in the normal and denervated skeletal muscle have been studied both histochemically as well as biochemically for a maximum period of 8 weeks of its postnatal development. In the normal muscle, a heterogenous population of fibres with respect to the enzyme distribution is observed. Relatively higher levels of enzyme in the denervated muscle and also the proliferation of extrafibrillar connective tissue in the diseased muscle show its specific association with the lytic processes.     

Effects of pyridoxal phosphate andl-dopapyridoxal phosphate on DOPA decarboxylase activity

Sommaire Les résultats obtenus démontrent une inhibition de l'activité de la DOPA decarboxylase par de haute concentration de PLP ou de faible concentration del-DOPA plus PLP. Ceci pourrait expliquer les observations cliniques et expérimentales précédentes démontrant que la pyridoxine antagonise l'effet del-DOPA utilisé dans le traitement de la maladie de Parkinson.     

Phosphoprotein phosphatase activity of bovine intestinal alkaline phosphatase

The phosphoprotein phosphatase activity of a commercial preparation of bovine intestinal alkaline phosphatase (EC 3.1.3.1) was examined using phosvitin and dentine phosphoprotein as substrates. Over 90% and 70% of the phosphorus from dentine phosphoprotein and phosvitin were hydrolyzed in 2 h. The optimum pH of the enzyme for the dephosphorylation of phosvitin and dentine phosphoprotein was nearly 6. No protein phosphatase activity was observed when the alkaline phosphatases from bovine liver and pulp were investigated.     

Role of phosphate and acid phosphatase during germination ofPinus pinea seed

Riassunto Durante la germinazione diPinus pinea i livelli di fosfato libero sono risultati essere correlati a quelli della fosfatasi acida, anche in differenti parti della plantula. I valori di fosfato totale sono costanti in tale periodo.

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This research was supported by a grant from the Impresa Enzimologica of the Italian Consiglio Nazionale delle Ricerche and from Ministero della Pubblica Istruzione.     

Tyrosine phosphatase activity in mitochondria: presence of Shp-2 phosphatase in mitochondria

Tyrosine phosphorylation by unidentified enzymes has been observed in mitochondria, with recent evidence indicating that non-receptorial tyrosine kinases belonging to the Src family, which represent key players in several transduction pathways, are constitutively present in mitochondria. The extent of protein phosphorylation reflects a coordination balance between the activities of specific kinases and phophatases. The present study demonstrates that purified rat brain mitochondria possess endogenous tyrosine phosphatase activity. Mitochondrial phosphatases were found to be capable of dephosphorylating different exogenous substrates, including paranitrophenylphosphate, ³²P-poly(Glu-Tyr)_4:1 and ³²P-angiotensin. These activities are strongly inhibited by peroxovanadate, a well-known inhibitor of tyrosine phosphatases, but not by inhibitors of alkali or Ser/Thr phosphatases, and mainly take place in the intermembrane space and outer mitochondrial membrane. Using a combination of approaches, we identified the tyrosine phosphatase Shp-2 in mitochondria. Shp-2 plays a crucial role in a number of intracellular signalling cascades and is probably involved in several human diseases. It thus represents the first tyrosine phosphatase shown to be present in mitochondria.Received 17 May 2004; received after revision 20 July 2004; accepted 26 July 2004     

Alkaline phosphatase activity in relation to DNA synthesis in synchronized hela S3 cells

Zusammenfassung In synchronisierten HeLa S3-Zellen wurde die Aktivität der alkalischen Phosphatase in Bezug auf den Zellteilungszyklus untersucht. Die geringste Aktivität wurde während der S-Phase und die höchste beim Minimum der DNS-Syntheseaktivität festgestellt, und es wird angenommen, dass die alkalische Phosphatase als Transphosphorylase zur Regulation der Polgrösse von Nucleotiden dient.

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Acknowledgment. This research was supported in part by a grant from the Israel Cancer Association.     

Semiquantitative estimation of phosphatase activity in the Sephadex layer

Zusammenfassung Rasche und einfache semiquantitative Methode zur Bestimmung der Aktivität von spezifischen Phosphatasen bei Reihenuntersuchungen: Detektion des Orthophosphats nach Diffusion des Enzyms durch eine Sephadex G-75-Schicht. Auf die Möglichkeiten ihrer Anwendung zur Bestimmung anderer Enzyme wird hingewiesen.

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The author is indebted to Mrs. J.Korytárová for technical assistance during this work.     

Acid phosphatase activity of cytolytic T cells during cytolysis].

The localization of Golgi areas, particularly lysosomal bodies detected by their acid phosphatase activities, has been determined in the cytotoxic lymphocytes conjugated to their target cell. The study points out the preferential situation of these organelles close to the contact area between the two cells during the target cell lysis.     

Possible role of intestinal alkaline phosphatase activity in thiamine transport

Summary Thiamine deficiency caused a marked decrease of intestinal alkaline phosphatase (al-Pase) activity, but had no effect on the Ca⁺⁺-ATPase activity and Ca⁺⁺-absorption in rats. The al-Pase activity was significantly decreased 1 h after oral administration of ethanol at 0.5 and 2.5 g/kg. In contrast, Mg⁺⁺-, Ca⁺⁺- and (Na⁺+K⁺)-ATPase activities did not change after the administration of ethanol. These findings show that the al-Pase activity, unlike the Ca⁺⁺-ATPase activity, is not related to Ca⁺⁺-absorption. A possible role of al-Pase activity in the active transport of thiamine in the intestine was discussed.     

Inorganic phosphate promotes relaxation of chemically skinned smooth muscle of guinea-pigTaenia coli

Summary In the presence of calmodulin and phosphate and an ATP-regenerating system, Triton-treated skinned fibers of theTaenia coli could be made to contract and relax by step changes of Ca⁺⁺ within about 30 sec. In the absence of phosphate, relaxation was slower, and during this slow relaxation tension was not maintained actively. The passive tension could be abolished by phosphate (3–6 mM). Phosphate had little effect on contractile tension but decreased the speed of contraction.Supported by the Deutsche Forschungsgemeinschaft. The excellent technical assistance of Miss Claudia Zeugner is acknowledged.     

Effect of exercise on ribonuclease activity in rat skeletal muscle

Summary Distribution of ribonuclease activity (measured at pH 7.6) in subcellular fractions of homogenates of rat skeletal muscle was investigated in sedentary animals and after 8 weeks running program. Training increased ribonuclease activity (expressed as units of enzyme per g of muscle protein). There was no increase in nuclear fraction, but in both cytoplasmic and mitochondrial fractions the RNA-ase activity increased 42% and 45% respectively.Acknowledgments. I would like to thank Prof. L. elewski and Dr J. Popinigis for helpful suggestions.     

Effect of benzimidazole on nicotinamide adenine dinucleotide phosphate phosphomonosterase activity in wheat leaves

Summary Nicotinamide adenine dinucleotide phosphate phosphomonoesterase was isolated and partially purified from wheat (Triticum aestivum L. var. Selkirk) leaves. The enzyme hadK _NADP value of 1.4×10^–4 M and a pH optimum of 5.9.In vitro activity of this enzyme was unaffected by precursors of NAD (nicotinamide and nicotinic acid) or cytokinis (kinetin and benzimidazole). However, when detached wheat leaves were treated with solutions of these compounds, the precursors lowered the specific activity while the cytokinins enhanced the activity. It is suggested that spatial separation and compartmentation of the enzyme and its substrate NADP account for the similar effect of benzimidazole on both.This work was supported by a grant No. A2698 from the National Research Council, Canada.