Phosphate from glycerophosphates

The acylation of dihydroxyacetone phosphate and the subsequent reduction of acyldihydroxacetone phosphate to monoacylphosphatidic acid provides an alternative route for phosphatidic acid synthesis. Acyltransferases which utilize dihydroxyacetone phosphate have been purified from several sources (Bell and Coleman, 1982). However, the relative contributions of dihydroxyacetone phosphate and glycerophosphate to phosphatidic acid synthesis remain controversial (O Doherty, 1978). 

However, the acid phosphatase activity of rat iiver lysosomes has recently been resolved into at least two enzymes [531]. Acid phosphatase is used in subcellular fractionation studies as a marker enzyme for lysosomes. Both acid phosphatase [E.C. 3.1.3.2] and alkaline phosphatase [E.C. 3.1.3.1] activities should not be confused with other specific phosphatases with high specificity requirements for substrate, e.g. glucose-6-phos-phatase, fructose-l,6-diphosphatase, phosphatidate phosphatase. Several assay procedures are available, u.v. estimation can be achieved using phosphoenolpyruvate as a substrate and lactate dehydrogenase in an indicator reaction [539]. Colorimetric assays can be based upon the liberation of phenol from phenylphosphate [540], upon the Uberation of phosphate from sodium /3-glycerophosphate [541], upon the hydrolysis of sodium phenolphthalein phosphate [542], or upon the hydrolysis of p-nitrophenyl phosphate [543]. 

It was Fujita and his colleagues (1969) who established the unique substrate specificity of -dependent p-nitrophenylphosphata e. For example, the enzyme did not hydrolyze -glycerophosphate or phenylphos-phate, which are the standard substrates for acid and alkaline phosphatases, nor did it hydrolyze ATP, AMP, 2 -(3 )-AMP, CMP, IMP, GMP, glucose-6-phosphate, or glucose-l-phosphate. From the noncompetitive nature of inhibition of both enzyme activities in the presence of ATP and p-nitrophenylphosphate, Fujita suggests diat there are two different active sites which may or may not be located on the same protein molecule. 

A different application of visible microscopy was pioneered by Gomori. In 1941 he showed that alkaline phosphatase could be specifically located by its hydrolysis of soluble phosphate esters (initially glycerophosphate). If calcium ions were present in the medium in which the sections were incubated, insoluble calcium phosphate precipitated as a result of the action of the hydrolase. The site of the precipitate could be visualized if cobalt or lead salts were subsequently added to replace calcium and the sections exposed to hydrogen sulfide. In principle many hydrolases and other enzymes could be studied using the appropriate substrates and precipitants. It was important to ensure that the products of the enzyme reactions did not diffuse from the sites where the enzymes were located. It was also essential that the reagents could reach the enzyme site. 

Chronic in vivo hemolysis produces serum lactic dehydrogenase elevations in patients with mitral or atrial valve cardiac prosthesis (J2). In a series of 11 such patients these increases ranged from 1.1 to 1.6 times the upper limit of normal (S29). Blood pH is altered in hemolyzcd specimens because carbonic anhydrase is liberated from the erythrocytes and presumably alters the distribution of H2CO3 and NaHCOs (B2). Hemolysis will effect acid phosphatase activity if the substrate is hydrolyzed by erythrocyte acid phosphatase. Thus, hemolysis would be of concern if phenyl phosphate was the substrate, but would have a negligible effect if )8-glycerophosphate, which is not hydrolyzed by red cell acid phosphatase, was used (Bl). 

Fig. 1. Prostatic acid phosphatase activity as a function of pH ( ) phenyl phosphate (O) p-nitrophenyl phosphate and (A) /8-glycerophosphate. Buffers Ac, acetate Cit, citrate and tris. From Nigam et al. (88).

Attack at C(2 ) in 3 -ribo-nucleolides is followed by a rapid release of phosphate as can be deduced from corresponding model systems such as the glycerophosphates (Samuni and Neta 1973 Steenken et al. 1974 Schuchmann et al. 1995) [reaction (276) k > 106 s-1 Schuchmann et al. 1995 see also Chap. 6.9],... 

Hypophosphites and phosphates, e.g. calcium hypophosphite and glycerophosphate, are used in considerable quantities as patent medicines and in medicine generally as accessory foods. Acid phosphates are used extensively in baking powders and various manufactured foods. The phosphorus requirements of the animal body are stated on p. 4, and also the supply of the element in certain vegetable products, while the probable role of phosphates in some biological processes is indicated on p. 169. From their intimate connection with life it will be gathered that by far the most important use of phosphorus compounds is in the manufacture of fertilisers (see Chap. XV.). 

The oxidation of L-glycerol 3-phosphate to dihydroxyacetone phosphate is catalyzed by two different enzymes. One is the cytoplasmic NAD-linked a-glycerophosphate dehydrogenase, and the other is the mitochondrial enzyme, which appears to contain flavin and iron. The latter enzyme was first studied by Green in 1936 (223). It was shown to be associated with respiratory particles, and widely distributed in animal tissues. The highest concentration of the enzyme was found in the brain. Lardy and co-workers (234) studied the enzyme in deoxycholate-solubilized particles obtained from skeletal muscle, confirmed the finding... 

The lysosomal and the soluble fractions of acid phosphatase were compared in several other ways. The values (milf) for the lysosomal fractions on various substrates were -glycerophosphate, 1.6 fructose 1,6-diphosphate, 2.0 p-nitrophenyl phosphate, 1.6 AMP, 0.43 they were not significantly different from those obtained with the soluble fraction. The pH-activity curves with these substrates were similar for the two fractions. Inhibition of phosphatase activity of the lysosomal and soluble fractions occurred at approximately the same concentration of fluoride or n- (+) -tartrate when j8-glycerophosphate, AMP, or fructose 1,6-diphosphate were used as substrates. However, with p-nitrophenyl phosphate... 

In addition to the studies cited above, there are several others showing that phenyl phosphate is much more readily hydrolyzed than j3-glycerophosphate by acid phosphatase from human erythrocytes, whereas no such marked difference exists with respect to human prostatic phosphatase (B2, Tl, T3). Unfortunately, there do not appear to be any systematic investigations of the substrate-velocity relationship for the acid phosphatases of other human tissues. In general, the available data would indicate that /3-glycerophosphate is a more specific substrate than phenyl phosphate for the detection and assay of acid phosphatase coming from the prostate. 

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