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Biochemical studies of testosterone 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) in rat submandibular gland (SMG) were performed. 14C-labeled testosterone or dihydrotestosterone (DHT) was incubated with subcellular fractions from rat SMG in the presence of 0.2 mM NADPH at 37 degrees C for 20 min in an atmosphere of 95% O2 and 5% CO2. Among the subcellular fractions, the high 5 alpha-reductase activity was detected in the nuclear fraction and 3 alpha-HSD in cytosol. Nuclear 5 alpha-reductase was efficiently solubilized in 2 mg digitonin per mg protein and 0.3 M KCl solution at 4 degrees C for 30 min. The maximum velocities (Vmax) of nuclear and solubilized 5 alpha-reductase activity for testosterone were 71.4 pmol/mg protein per min and 25.4 pmol/mg protein per min. Apparent Michaelis constant (Km) of nuclear and solubilized enzymes for testosterone were calculated as 11.1 microM and 16.7 microM by the Lineweaver Burk plot, respectively. The activity of solubilized 5 alpha-reductase from nuclei was stable by NADPH and KCl, and the molecular weight of the enzyme was estimated as 158 K.D approximately 200 K.D by Bio-Gel A-1.5 m column chromatography. The column chromatography also showed a peak of 3 alpha-HSD activity in cytosol, revealing the molecular weight of approximately 50 K.D. However, the elution peak of the 3 alpha-HSD was effectively decreased by KCl in Tris-HCl buffer. The molecular weight of 5 alpha-reductase and 3 alpha-HSD in SMG were similar to those in prostate. A stable and extractable 5 alpha-reductase was demonstrated in nuclei of rat SMG with possessing a considerable affinity for testosterone and also high 3 alpha-HSD activity for DHT was revealed in cytosol of the tissue.

Abstract:Changes in the secretory proteins of the rat submandibular saliva and its amino acid composition, brought about by a single dose of the parasympathomimetic drug, Pilocarpine, were studied.After collecting unstimulated saliva, the secretion was elicited by Pilocarpine and the saliva was collected at 10-minute intervals.Unstimulated saliva could be separated into seventeen components by SDS-electrophoresis, the stimuli were separated into twenty-six components both at 1 hr and 2 hr. The carbohydrate-protein complex of stimulated saliva observed eleven components in contrast with unstimulated saliva which had eight components.Both unstimulated and stimulated saliva were consisted of aspartic acid, glutamic acid and proline-rich residues and with a given stimuli, the amino acid composition had a tendency to increase the basic amino acid residues.The low molecular bands in electrophoretogram contained in the cytosol fraction of the submandibular gland were absent from stimulated cytosol at 1 hr and 2 hr, and Pilocarpine administration was effective in increasing the high molecule proteins in the cytosol at 1 hr and 2 hr.These results suggested that Pilocarpine brings about the discharge of salivary granule components into saliva and the differences in the secretory components with Pilocarpine stimuli and unstimuli, were evidenced.

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