K. C. Tsou

Fluorometric assay of serum 5′-nucleotide phosphodiesterase in normal humans and cancer patients

Abstract A sensitive method of assaying serum 5′-nucleotide phosphodiesterase (PDase) is described. A preliminary study did not find significant differences between the levels of PDase in cancer patients and those of normal human sera.

A new colorimetric method for the determination of alkaline phosphatase with indoxyl phosphate

Abstract A new colorimetric method for alkaline phosphatase has been developed with indoxyl phosphate as a chromogenic substrate. The indoxyl liberated by enzymic hydrolysis is oxidized to the blue indigo in presence of air. The measurement of the colored dye is made possible by the use of polyvinyl alcohol as a protective colloid. Kinetic study of this substrate supported most of its known advantages for histochemical purposes. Free formaldehyde, when present, inhibits the oxidation, but formaldehydereacted alkaline phosphatase remains active for this substrate.

Preparation, properties, and uses of two fluorogenic substrates for the detection of 5′-(venom) and 3′-(spleen) nucleotide phosphodiesterases

Abstract The synthesis of two new substances useful for the detection of phosphodiesterases by the zymogram method is described. The compounds are 4-methylumbelliferyl phosphate esters of thymidine, made by condensation of the appropriate phosphomonoester and alcohol in the presence of dicyclohexylcarbodiimide. It is shown that 4-methylumbelliferyl thymidine 5′-phosphate is preferentially cleaved by phosphodiesterase I and the equivalent 3′-phosphate by phosphodiesterase II. Zymograms which have been stained with these compounds reveal only the appropriate enzyme; phosphomonoesterase does not appear on the gels.

ON THE USE OF POTASSIUM FERRICYANIDE-FERROCYANIDE WITH 5-BROMOINDOXYL ACETATE IN THE HISTOCHEMICAL DEMONSTRATION OF ESTERASE

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Fluorometric assay of 2′, 3′ -cyclic adenosine monophosphate 3′-phosphohydrolase with 1,N6-etheno-2-aza-adenosine 2′, 3′ -monophosphate1

THE PRESENCE of 2’,3’cyclic adenosine monophosphate 3’phosphohydrolase (CNPase) in the central nervous system (CNS) was first reported by DRUMMOND el al. (1962). Subsequent studies by KURIHARA & TSUKUDA (1967) showed that the enzyme is associated with the myelin sheath of the CNS. Since then, CNPase has been widely used as a marker enzyme for the isolation of myelin or for the estimation of myelin contamination in cell fractionation (NORTON, 1971 ; DMONTE er al, 1971 ; QUARLES et al, 1972). Current assay methods for CNPase include (a) isolation of the product (2’-adenosine monophosphate) by paper electrophoresis (DRUMMOND rt al, 1962), paper chromatography (KURIHARA & TSUKUDA, 1967), or thin layer chromatography ( Z A N E ~ A et al, 1972; WAEHNELDT & MANDEL, 1972) and determination of the product concentration spectrophotometrically ; (b) dephosphorylation of 2-adenosine monophosphate with alkaline phosphatase in a second incubation and determination of the liberated inorganic phosphate (OWSON er al, 1969); and (c) fluorometric determination of residual substrate, l,Nb-etheno-adenosine-2.3cyclic monophosphate after precipitation of the product by Ba(OH),-ZnS04 (TRAMS, 1973). Methods (a) and (b) are tedious or insensitive. Method (c), while sensitive and elegant, is not accurate for low enzyme activity in our hands. Recently, we have synthesized fluorescent adenosine derivatives ( 1,N6-etheno-2-aza-adenosine derivatives) which have an excitation maximum of 358 nm and an emission maximum of 495 nm (YIP & Twu, 1973; Tsou et al, 1974b). The fluorescent characteristics of these compounds enable their use in histochemistry and cytochemistry. In addition, some of these analogs are efficient substrate for enzymes which normally act on adenosine derivatives. For example, the effficiency of the hydrolysis of 1,N6-etheno-2-azaadenosine 3,Scyclic monophosphate by beef heart 3,scyclic adenosine monophosphate phosphodiesterase is about 75% that of the parent adenosine compound (Tsou er d. 1974a). This paper reports the successful synthesis of 1 .N6-etheno-2-aza-adenosine-2’,3’cyclic monophosphate (Fig. I ) and its usefulness for the assay of CNPase.

1,N6-etheno-2-aza-adenosine 3′,5′-monophosphate: A new fluorescent substrate for cycle nucleotide phosphodiesterase

Abstract 1, N 6 -etheno-2-aza-adenosine 3′,5′-monophosphate (cyclic 2-aza-ϵ-AMP) has been shown to be a sensitive and an efficient substrate for the assay of cyclic-nucleotide phosphodiesterase. The relative activity is 75% compared to cyclic AMP. Two K m values of 503 and 15 μ m were observed with the beef heart enzyme.

A study of yeast α-galactosidase with naphthyl α-d-galactopyranosides as chromogenic substrates

Abstract The mechanism of α-galactosidase action was studied kinetically. A comparison of the rate of their enzymic hydrolysis shows 6-bromo-2-naphthyl->1-naphthyl->2-naphthyl-α- d -galactopyranosides. Such data suggest the influence of the aglycone group in the α- d -galactosides is that of a steric acceleration. K m and V max for the three naphthyl α- d -galactopyranosides were obtained. Mercuric acetate was found to be an inhibitor for α-galactosidase whereas galactose, glucose, fructose, lactose, galacturonic acid, cysteine, histidine, imidazole, and aniline were found to be noninhibitors. Certain metal cyanides were found to be activators.

Letters to the editor: A new method for adenosine triphosphatase adaptable for fluorescent and electron microscope application.

therefore also been accomplished with the lead trapping method. In spite of the wide application of these light and electron microscope methods, it is commonly acknowledged that certain limitations and artifacts are unavoidable. Perhaps the most serious ones are that lead ion is an inhibitor of ATPase and that there is a lack of specificity of the reaction in that other than APT, both guanosine triphosphate and cytidine tn-