Changes in pools of acid-soluble phosphorus compounds induced by phosphorus starvation in Neurospora.
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1. A method is described for the extraction, purification and separation of acid-soluble phosphorus compounds from mammalian semen. [8-(14)C]ATP and [8-(14)C]AMP were used as internal recovery standards to measure the breakdown and loss of these nucleotides in the procedure. 2. Bull, ram, boar and stallion semen was separated into seminal plasma and spermatozoa and the two fractions were examined separately. The overall composition of the mixture of the phosphorus compounds extracted from the two fractions was similar for the four species. 3. Glycerylphosphorylcholine and glycerylphosphorylinositol were the two phosphorus compounds identified in extracts of seminal plasma. ATP, ADP, AMP, GTP, GDP, NAD, fructose 1,6-diphosphate and glucose 6-phosphate were identified in extracts prepared from spermatozoa.
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Patients with hypophosphatasia caused by a deficiency of alkaline phosphatase first showed marked accumulation of phosphoethanolamine and other phosphorus compounds in kidney and liver, while in placenta and intestine contents of these compounds were within a normal range. Furthermore, 32P-incorporation in cultured skin fibroblasts of patients with hypophosphatasia was increased about two to three times of control. FPLC chromatographic analysis also indicates that the accumulated phosphorus compounds in hypophosphatasia was smaller molecular phosphorus containing compounds. These data provide new pathophysiological aspect of hypophosphatasia.
A single-paper chromatographic system has been developed, capable of resolving Tc-99m dioxide, [99mTc] pertechnetate and Tc-99m phosphorus compounds. The best separations are obtained with CM82 paper developed in 0.5 m naCl, and 3MM or ashless No. 40 paper developed in 1 M sodium acetate buffer. In these systems, 99TcO2 remains at the origin, while 99mTcO4--and Tc-99m phosphorus compounds move with Rf values of 0.56--0.75 and 0.80--1.0, respectively.
Pulsed-gradient 31P NMR was used to measure the diffusion rates of phosphorus compounds in aqueous solution and in living muscles. The diffusion rates of creatine phosphate and inorganic phosphate in intact frog muscle cells were reduced by a factor of approximately 2 from those in aqueous solution, which suggests that the apparent intracellular viscosity is approximately 2 times larger than in aqueous solution.
Structural requirements of inorganic phosphorus compounds as specific activators or inhibitors for phospholipase A2 and phospholipase B were investigated using orthophosphate, pyrophosphate and polyphosphate. It was observed that orthophosphate and pyrophosphate stimulated the activities of phospholipase A2 from bee venom, snake (Naja naja) venom and pig pancreas, and also phospholipase B from the yeast Torulaspora delbrueckii. However, polyphosphate was found to act as an inhibitor for phospholipase A2 in the above species and also for phospholipase B from T. delbrueckii. Orthophosphate and pyrophosphate induced gradual aggregation of liposome, but polyphosphate prolonged the lifetime of the liposome, suggesting that orthophosphate and pyrophosphate destabilize the bilayer structure of phosphatidylcholine and polyphosphate stabilizes it.
Metabolism of macroergic phosphorus compounds was studied in high- and low-productive isogenic strains of Penicillium chrysogenum. It was shown that the levels of the high-polymer polyphosphates (fractions PP1, PP2 and PP3) in the strain intensively producing penicillin were 2-3 times higher than those in the low-productive strain by the 2nd day of the fermentation process (the period of penicillin production). The levels of pyrophosphate and ATP in the mycelium during the fermentation process did not significantly differ in the strains. The study on the relation between metabolism of the high-molecular polyphosphates and conditions of the culture growth and antibiotic production revealed that their accumulation was connected with biosynthetic processes giving rise to the growth of P. chrysogenum, while their consumption with penicillin production. The dynamics of the pyrophosphatase and polyphosphatase activity in the mycelium of the strains was studied.
The dependency of fluid secretion on extracellular Na+ and the levels of phosphorus compounds were studied in the perfused canine mandibular gland (using 31P-NMR). During control perfusion, the resting levels of creatine phosphate (CP) and ATP were 0.62 +/- 0.05 mmol . kg-1 gland and 0.42 +/- 0.04 mmol . kg-1 (mean +/- S.E., n = 9), respectively. Acetylcholine (Ach; 1 mumol . l-1 for 3 min) induced a salivary secretion and decreased the CP level. When Na+ in the perfusate was completely replaced with Li+, Ach induced only a minimal salivary secretion and no change in the ATP and CP levels. Restitution of Na+ to the perfusion, even without added Ach, caused a decrease in ATP and CP, and a small increase in salivary secretion. These results suggest that the activity of Na+/K+ ATPase is increased inversely via a rise of the intracellular Na+ concentration and that the salivary secretion is induced not only by added secretagogues but by an increase in the Na+ entry without added secretagogues.
Responses of beta-adrenoceptor (beta-AR) in rat soleus to gravitational unloading and/or changes in the levels of phosphorus compounds by feeding either creatine or its analogue beta-guanidinopropionic acid (beta-GPA) were studied. A decrease in the density of beta-AR (about -35%) was induced by 10 days of hindlimb suspension, but the affinity of the receptor was unaffected. Suspension unloading tended to increase the levels of adenosine triphosphate and phosphocreatine and decrease inorganic phosphate. Even without unloading, the beta-AR density decreased after an oral creatine supplementation (about -20%), which also tended to elevate the high-energy phosphate levels in muscle. However, an elevation of beta-AR density was induced (about +36%) after chronic depletion of high-energy phosphates by feeding beta-GPA (about +125%). Data suggest that the density of beta-AR in muscle is elevated if the high-energy phosphate contents are chronically decreased and vice versa. However, it may not be directly related to the degree of muscle contractile activity.
Simultaneous determination of 32P and 33P radioactivities was carried out with the liquid scintillation counter (Aloca LSC 670). For the quenching correction, the channel ratio method was used, and the proper gain and window level were set to get the maximum performance number or Klein-Eisler value. Using this method the radioactivities of the 32P and 33P double labelled compounds which were separated with paper chromatography were determined. New index, RSA (relative specific activity, i.e. 32P/33P) was proved to be effective as well as specific activity for the studies of the transport and metabolism of phosphorus compounds in the plant roots.
Phosphorus nuclear magnetic resonance (31P NMR) spectroscopy was used to study energy supply for protein secretion in the isolated perfused rat pancreas. Stimulation with cholecystokinin (CCK-8) increased fluid secretion and protein output. With 10pM of CCK-8, the tissue contents of ATP, inorganic phosphate (Pi), and creatine phosphate (PCr) remained unchanged. With 100pM of CCK-8, which induced the maximum response in fluid secretion and protein output, Pi increased slightly, ATP and PCr remained almost unchanged. A high dose of CCK-8 (1 nM) suppressed the fluid and protein secretory rates, decreased ATP, and increased Pi significantly, but PCr showed a tendency of increase. Significant changes in ATP and Pi occurred on withdrawal of CCK-8 (1 nM), suggesting activation of ATP hydrolysis for recovery from secretory suppression. During stimulation with 0.1, 0.3, and 3.0 microM of acetylcholine, the similar dose-dependent response was observed in the secretion and the phosphorus metabolism. The present study demonstrated that cytosolic energy metabolism for secretory responses in the pancreas is low and the Lohmann reaction showed less contribution than in the salivary gland. The findings suggest that the energy supply for protein secretion may be cytosolic diffusion of ATP and that the Lohmann reaction system may contribute to synthesis and storing of secretory protein at resting state.
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Trivalent oxygenated phosphorus ligands include alkyl and aryl phosphites, (RO)3P, phosphonites, (RO)2PR, and phosphinites, ROPR2. All such compounds tested, with the exception of triphenyl phosphite, interact with ferrous cytochrome P-450 and its denatured form, cytochrome P-420, to produce complexes having two peaks in the Soret region of their optical difference spectra. Careful evaluation of these spectra indicate that they arise for different reasons for each of the two cytochromes. Clear evidence shows that cytochrome P-450 is not denatured by these ligands. The high affinity of these ligands for heme iron is indicated by small Ks values. The experimental results are used to substantiate a theory of the origin of microsomal double Soret spectra and the nature of the environments available for microsomal cytochromes P-450 and P-420.
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