Hiroyasu Nakata

Purification and characterization of phenylalanine 4-monooxygenase from rat liver.

Phenylalanine 4-monooxygenase (L-phenylalanine, tetrahydropteridine:oxygen oxidoreductase (4-hydroxylating), EC 1.14.16.1) was purified approx. 600-fold to apparent homogeneity with a 48% yield from rat liver. Two distinct active forms were separable by calcium phosphate gel chromatography and numbered based on their order of elution from the gel column. The predominant form, Form I, had an estimated molecular weight of about 240 000. The enzyme gave a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis, the molecular weight of which was estimated to be approx. 51 000, indicating that the enzyme might be composed of four identical subunits. The molecular properties of Form I were: sedimentation coefficient, 10.1 S; Stokes radius, 55 A degrees; diffusion coefficient, 3.90 x 10(-7) cm2/s; frictional ratio, 1.33 and isoelectric point, pH 5.6. The enzyme contained approx. 0.6 mol of iron and 0.3 mol of phosphate/mol of subunit of the enzyme. No significant differences in kinetic properties of the two forms, Form I and Form II, were observed. Amino acid analysis studies revealed that the amino acid composition of Form I was essentially identical with that of Form II, indicating that both forms might be the products of the same gene. There were, however, minor differences in the phosphate content and the isoelectric point between the two forms.

Solubilization and partial characterization of adenosine binding sites from rat brainstem.

Binding sites for adenosine were solubilized from rat brainstem membranes with either sodium cholate, sodium deoxycholate or 3‐[3‐cholamidopropyl)dimethyl‐ammonio]‐1‐propanesulfonate. About 30% of the binding activity were released by these detergents as assayed by [3H]phenylisopropyladenosine (PIA) binding. Specific [3H]PIA binding to the solubilized fraction was saturable and was found to be a monophasic saturation profile. In contrast, [3H]PIA binding sites. By gel filtration on a Sepharose CL‐6B biphasic profile suggesting the presence of two binding sites. By gel filtration on a Sepharose CL‐6B column, the adenosine binding site—detergent complex was estimated to have app. M r 280 000 and r s = 5.4 nm.

Development of an antiserum to rat-brain A1 adenosine receptor: Application for immunological and structural comparison of A1 adenosine receptors from various tissues and species

An antiserum was developed in a rabbit against rat-brain A1 adenosine receptor. This antiserum recognized the denatured form of the purified rat-brain A1 adenosine receptor in immunoblot analysis and the native form of the receptor in the immunoprecipitation analysis. Immunoblot analysis of unpurified or purified adenosine receptor preparations from rat-brain membranes revealed a major immunoreactive band at a position of molecular mass of approx. 35 kDa, which corresponds to the position of purified rat-brain A1 adenosine receptor. Although A1 adenosine receptors from other rat tissues such as testis and adipocyte were also found to be immunoreactive with this antiserum by immunoblot analysis, purified human-brain A1 adenosine receptors showed a poor reactivity with this antibody. The order of the relative immunoreactivity of these A1 adenosine receptors with the antiserum was found to be brain > adipocyte > or = testis. Moreover, the immunoreactivity of these receptors significantly increased after these receptor preparations were deglycosylated by endoglycosidase F. After the deglycosylation, no significant differences in both the immunoreactivity and molecular mass among these receptor preparations were found on the immunoblot. These results suggest that the differences in the molecular mass or immunoreactivity among the A1 adenosine receptor preparations from three rat tissues were mainly due to the difference of sugar moiety present in each receptor molecule. These data are the first to provide analyses of immunological characteristics of A1 adenosine receptors from different tissues and species.

Role of Calmodulin in Neurotransimitter Syntheis

Tyrosine 3-monooxygenase (EC 1.14.16.2) and tryptophan 5-monooxygenase (EC 1.14.16.4) are generally believed to be the rate-limiting enzymes in the biosynthesis of the neurotransmitters, catecholamines and serotonin, respectively, and therefore the regulation of their activities is of particular importance. At least three calmodulin-dependent protein kinases differed in their molecular weights and substrate specificities, designated I, II, and III in the order of decreasing molecular weight, in rat brain cytosol. Among them, calmodulin-dependent protein kinase II with a molecular weight of about 540,000 appeared to occur only in the nervous tissues. Kinase II was found, on the one hand, to phosphorylate tyrosine 3-monooxygenase and tryptophan 5-monooxygenase, leading to the activation of these monooxygenases in the presence of activator protein and, on the other hand, to phosphorylate tubulin and microtubule-associated protein 2, which results in disassembly of the microtubules that had been assembled. These results suggest the possibility that both the secretion and biosynthesis of monoamine neurotransmitters stimulated by Ca2+ influx in the nervous system may be regulated by calmodulin-dependent protein kinase II via the phosphorylation of microtubule proteins and the phosphorylation of the monooxygenases that are the rate-limiting enzymes in the biosynthesis of the neurotransmitters.

Studies on the reaction intermediate of protocatechuate 3,4-dioxygenase Formation of enzyme-product complex

The nature of the oxygenated intermediate observed (Fujisawa, H., Hiromi, K., Uyeda, M., Okuno, S., Nozaki, M. and Hayaishi, O. (1972) J. Biol. Chem. 247, 4422--4428) during the reaction of protocatechuate 3,4-dioxygenase (protocatechuate:oxygen 3,4-oxidoreductase (decyclizing), EC 1.13.11.3) was investigated. 3,4-Dihydroxyphenylpropionic acid and 3,4-dihydroxyphenylacetic acid were used as substrates of the enzyme to slow down the rate of the reaction. The enzyme reactions were performed under conditions where the concentration of the organic substrate was lower than those of the enzyme and oxygen in the reaction mixture. The reactions were stopped before completion by the addition of hydrochloric acid or guanidine hydrochloride and then the organic compounds were extracted from the reaction mixture to be analyzed. The qualitative analyses by thin-layer chromatography revealed that there was no species other than the organic substrate and the enzymatic reaction end-product during reaction. The quantitative spectrophotometric analyses revealed that the organic substrate which had participated in the formation of the oxygenated intermediate existed as a species indistinguishable from the reaction end-product, indicating that the oxygenated intermediate was not a simple complex of oxygen, substrate and the enzyme, i.e., a ternary complex, but a species rather close to a binary complex of product and the enzyme.

A simple assay of solubilized adenosine receptors with nitrocellulose membrane filters.

A rapid and simple assay of solubilized adenosine receptors with nitrocellulose membrane filters is described. This assay was sensitive and reproducible when applied to adenosine receptors solubilized from rat brainstem membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Appropriate values of dissociation constants for the solubilized adenosine receptors to their tritiated agonists were obtained by the membrane filter technique. This method should be applicable for the assay of a variety of solubilized receptors.