The study of physiological chemistry on active fragments of the salivary gland hormone

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We previously presented evidence that the salivary gland hormone, parotin, is composed of a subunit which has a molecular weight of 45000 and possesses the biological and immunological activities of parotin. In this paper the isolation of fragments from a tryptic digest of the subunit and their physiological chemistry were investigated to obtain active components of the subunit.After 2 g of the subunit were digested with 20 mg of trypsin for 20 hours at 37°C, the tryptic digest was chromatographed on a Sephadex G-25 column equilibrated with 0.1 M NH4HCO3. The eluate was fractionated into 9 parts from Fr. A to Fr. I in order of their molecular size. An increasing activity in the rabbit-circulating leucocyte number of the subunit was separated into 4 fractions of Fr. A. B, D and E. Rabbit serum Ca decreased and immunological activities were recovered into only Fr. H and Fr. A, respectively. Leucocyte increase and immunological activities of Fr. A were concentrated by successive chromatographies with Sephadex G-50 and QAE-Sephadex A-25 column. A purified component, Fr. AA-1, was found to have a molecular weight of 9100 by 15% polyacrylamide gel electrophoresis in 0.1% sodium dodecyl sulfate, N-terminal amino acid sequence of Leu-Tyr-Ile-Leu-Tyr-Phe-Phe-Glx-by combined dansyl-Edman procedure, C-terminal amino acid sequence of-Ile-Val-Leu-Leu-Lys (OH) by carboxypeptidase A + B method and glycopeptide consisting of 6.9% amino sugar and 7.4% uronic acid. A Ca decreasing active principle, Fr. H-1, purified from Fr. H by paper chromatography, was characterized to be pentapeptide of Asp-Tyr-Glu-Trp-Lys (OH). Both Fr. AA-1 and Fr. H-1 were not positioned on N-and C-terminals of the subunit molecule.Whole-body autoradiographies of the subunit and active components labeled with 131I in mice revealed that the subunit and Fr. AA-1 specifically were localized in the bone in addition to the liver, kidney, thyroid, spleen, adrenal and submaxillary gland, but Fr. H-1 did not distribute to the bone.