The uptake and metabolism of [32P]pyrophosphate by mouse calvaria in vitro

In order to study the uptake and metabolism of PP(i) by bone, (32)PP(i) was added to the medium surrounding explanted mouse calvaria maintained in organ culture. Most of the PP(i) was hydrolysed during incubation, but there was a measurable entry of intact PP(i) into bone. When (32)P(i) was added to the medium, synthesis of PP(i) and organic phosphates from P(i) was observed in bone. There was no detectable passage of PP(i) from bone into the medium. These results are discussed in terms of two models of pyrophosphate hydrolysis and exchange. Some quantitative estimates about the fate of PP(i) in bone were made.     

PHosphate metabolism and foetal growth in the rat. I. Net transfer of 31P and 32P phosphate from the maternal plasma to the normal placenta

Net transfer of 31P and 32P inorganic phosphate form the maternal plasma to the rat chorio-allantoic placenta has been studied after intraperitoneal injection of [32P] ortho-phosphate in primigravid females at the 12th day or late stages of gestation. The concentration and label uptake per unit weight of placenta of the inorganic phosphate (Pi), organic-bound acid-soluble phosphate (POAS) and organic-bound acid-insoluble phosphate (POAIS) fractions are negatively correlated with increasing placental weight, whereas their specific activities are independent of placental weight. The amount and label uptake per whole placenta of the Pi, POAS and POAIS fractions are positively correlated with increasing placental weight. The placental concentrations of inorganic phosphate and calcium are positively related without, however, any marked accumulation of calcium. The growing placenta is thus shown to reduce progressively, on a unit weight basis, both the inorganic phosphate uptake from the maternal plasma and its further incorporation into organic-bound fractions. There is no evidence of a control by the foetal weight, acting per se, on these placental activities.     

Phosphate metabolism and foetal growth in the rat. II. Net transfer of 31P and 32P inorganic phosphate from the maternal plasma to the normal foetus

Net transfer of 31P and 32P inorganic phosphate from the maternal plasma to the rat foetus has been studied after intraperitoneal injection of [32P] ortho-phosphate in primigravid females at the 12th day or later stages of gestation. The concentration per unit weight of foetus of the inorganic phosphate (P1) fraction increases markedly with increasing foetal weight; labelling data [inverse relationship between P1 concentration and specific activity, absence of precursor/product relationship between P1 and acid-soluble organic-bound phosphates (POAS)] show this increase to result in part from the formation of a relatively inert metabolic pool, presumably in mineralized tissue. The foetal concentrations of calcium and inorganic phosphate show a strong positive correlation, both increasing markedly with foetal weight. The progressive accumulation of calcium does not, however, account entirely for the rising concentration of inorganic phosphate. The concentration per unit weight of foetus of the POAS fraction remains stable for foetuses smaller than 2 000 mg. In heavier foetuses (greater than 2 000 mg) the POAS concentrations are, with an abrupt transition, distinctly lower, rising however slightly with increasing foetal weight. The concentration per unit weight of foetus of the acid-insoluble organic-bound phosphate (POAIS) fraction decreases slightly with increasing foetal weight. The label uptake per unit weight of foetus of both POAS and POAIS fractions is negatively correlated with increasing foetal weight. The amount and label uptake per whole foetus of the P1, POAS and POAIS fractions are positively correlated with increasing foetal weight. Phosphate transfer to the foetus increases continuously, being maximal at or near birth.     

Biochemical aspects of the neuroprotective mechanism of PTEN-induced kinase-1 (PINK1)

Mutations in PTEN-induced kinase 1 (PINK1) gene cause PARK6 familial Parkinsonism. To decipher the role of PINK1 in pathogenesis of Parkinson's disease (PD), researchers need to identify protein substrates of PINK1 kinase activity that govern neuronal survival, and establish whether aberrant regulation and inactivation of PINK1 contribute to both familial Parkinsonism and idiopathic PD. These studies should take into account the several unique structural and functional features of PINK1. First PINK1 is a rare example of a protein kinase with a predicted mitochondrial-targeting sequence and a possible resident mitochondrial function. Second, bioinformatic analysis reveals unique insert regions within the kinase domain that are potentially involved in regulation of kinase activity, substrate selectivity and stability of PINK1. Third, the C-terminal region contains functional motifs governing kinase activity and substrate selectivity. Fourth, accumulating evidence suggests that PINK1 interacts with other signaling proteins implicated in PD pathogenesis and mitochondrial dysfunction. The most prominent examples are the E3 ubiquitin ligase Parkin, the mitochondrial protease high temperature requirement serine protease 2 and the mitochondrial chaperone tumor necrosis factor receptor-associated protein 1. How PINK1 may regulate these proteins to maintain neuronal survival is unclear. This review describes the unique structural features of PINK1 and their possible roles in governing mitochondrial import, processing, kinase activity, substrate selectivity and stability of PINK1. Based upon the findings of previous studies of PINK1 function in cell lines and animal models, we propose a model on the neuroprotective mechanism of PINK1. This model may serve as a conceptual framework for future investigation into the molecular basis of PD pathogenesis.     

Phosphate metabolism and foetal growth in the rat. III. Net transfer of 31P and 32P inorganic phosphate from the maternal plasma to the placenta after foetectomy

In primigravid rat females at the 12th day or later stages of gestation one foetus is surgically removed and the amniotic sac allowed to retract freely around the remaining placenta in situ. One hour to 10 days after foetectomy [32P]ortho-phosphate is injected intraperitoneally. The post-foetectomy placenta and matched normal placenta and foetus in the contralateral horn are taken after labelling periods of one or three hours and analysed for calcium, inorganic phosphate, organic-bound acid-soluble and organic-bound acid-insoluble phosphates in their [31P] and [32P] forms. The acutely foetectomized placenta--up to 24 hours after surgery--may serve as an appropriate model of the physiologically functioning syncytiotrophoblast: it is indeed noteworthy that organic-bound phosphate placental concentrations and labelling are little affected in the early post-foetectomy stages. On the other hand the placental inorganic phosphate concentration and labelling are profoundly affected by foetectomy. A surplus radioactive incorporation in the Pi fraction is observed which is strongly related to the weight of the control foetus and may represent up to twelve times the label incorporation in the control placenta; it decreases as the time interval since foetectomy lengthens. A cumulative increase in Pi concentration is observed which is strongly related to calcium concentration and may reach twenty-five times the maternal plasma Pi concentration. The presence of two independent pools of Pi is suggested: one geared to the metabolic needs of the placenta itself, the other representing the Pi normally transported to the foetus under metabolic control by the latter. The possibility of an electroneutral PO4(2-)/Ca2+ co-transport is evoked.     

Membrane lipids composition and metabolism during early embryonic development. Phospholipid subcellular distribution and 32P labeling

Phospholipid composition and 32P metabolism were studied in oocytes and early developing embryos of the toad, Bufo arenarum, Hensel. The content and distribution of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, sphingomyelin, phosphatidylserine, and diphosphatidylglycerol in embryos, whole oocytes, and the subcellular fractions of both were determined. Phosphatidylcholine and phosphatidylethanolamine were the major constituents of yolk platelet. Diphosphatidylglycerol was confined to the mitochondrial fraction, where it represented about 7% of the total phosphoacylglycerols. Relatively large amounts of sphingomyelin were found in microsomal and postmicrosomal supernatants. After in vivo labeling with 32P, the early development of individual phospholipids in subcellular fractions and in whole eggs was followed. The greatest uptake was found in mitochondrial and yolk platelet fractions. A steady increase in the amount of 32P present in phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol was seen in the whole embryo from oocyte to late gastrula stage and in all subcellular fractions. Phosphatidic acid exhibited a slight decrease in specific activity, except in the yolk platelet fraction. This high 32P incorporation would indicate a rapid and uneven polar headgroup turnover determined by phospholipid class and subcellular fraction. At the same time, the phospholipid content of the subcellular fractions studied remained unchanged during early embryogenesis. Moreover, 32P was actively incorporated into the individual phospholipids in the absence of measurable net synthesis.     

Synthesis P1-dolichyl P2-alpha-D-mannopyranosyl pyrophosphate. The acid and alkaline hydrolysis of polyisoprenyl alpha-D-mannopyranosyl mono- and pyrophosphate diesters.

P1-Dolichyl P2-ALPHA-D-mannopyranosyl pyrophosphate (9) has been chemically synthesized by a method developed for the corresponding citronellyl derivative, which also contains a saturated alpha isoprene residue. In each case, the P1-polyisoprenyl P2-diphenyl pyrophosphate was treated with 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl phosphate to give a fully acetylated pyrophosphate diester, which was purified chromatographically and subsequently deacetylated. The citronellyl and dolichyl pyrophosphate diesters were compared with the previously synthesized citronellyl and dolichyl alpha-D-mannopyranosyl phosphate, respectively, by chromatography and by hydrolysis experiments. Good separations of the monophosphate from the corresponding pyrophosphate were achieved by silica gel tlc in a variety of solvent systems. Brief dilute acid hydrolysis of both the mono- and pyrophosphate diesters gave D-mannose and no alpha-D-mannosyl phosphate, the other products being polyprenyl phosphate and pyrophosphate, respectively. When the polyprenyl alpha-D-mannopyranosyl mono- and pyrophosphate diesters were treated with hot dilute alkali, the major products were polyprenyl phosphate and substances arising from the breakdown of D-mannose, indicating that the alpha-D-mannosyl phosphate bond was the most labile linkage in both compounds. However, the formation of a small proportion of free dolichol indicated that alpha-D-mannosyl phosphate was also formed to a minor extent. The interpretation of the results of the alkaline hydrolysis was complicated by the instability of D-mannose under basic conditions, it being almost completely degraded by even a brief treatment.     

6-phosphofructokinase (pyrophosphate). Properties of the enzyme from Entamoeba histolytica and its reaction mechanism.

The inorganic pyrophosphate-requiring 6-phosphofructokinase of Entamoeba histolytica has been further investigated. The molecular weight of the enzyme is approximately 83,000 and its isoelectric point occurs at pH 5.8 to 6.0. The divalent cation requirement for reaction was explored. In the direction of fructose 6-phosphate formation half-maximal rate required 500 muM magnesium ion; in the direction of fructose bisphosphate formation 8 muM magnesium ion sufficed. ATP, PPi, polyphosphate, acetyl phosphate, or carbamyl phosphate cannot replace PPi as phosphate donor for the conversion of fructose 6-phosphate to fructose bisphosphate. In the direction of fructose 6-phosphate formation arsenate can replace orthophosphate. Isotope exchange studies indicate that little or no exchange occurs between Pi and PPi or between fructose 6-phosphate and fructose bisphosphate in the absence of a third substrate. These findings appear to rule out phosphoenzyme formation and a ping-pong reaction mechanism. PPi, Pi, and fructose bisphosphate are competitive inhibitors of fructose bisphosphate, PPi, and fructose 6-phosphate, respectively. This argues against an ordered mechanism and suggests a random mechanism. Fructose 6-phosphate and Pi were noncompetitive with respect to each other indicating the formation of a dead end complex. These product inhibition relationships are in accord with a Random Bi Bi mechanism.     

The influence of the fungicide folcidin on the distribution and metabolism of32P in gherkin plants

High accumulation of³²P was observed in the leaves of intact gherkin plants 9 days after their roots had been treated with 0.005% suspension of the systemic fungicide Folcidin 50WP (cypendazole),i.e. 8 days after the roots had been exposed to labelled phosphate. Folcidin also influenced phosphorus metabolism in the plants. High biologic cytokinin-like activity of the fungicide was established when using a callus cytokinin bioassay.