ABSTRACT
The regulatory peptides Phk13 (301-327) and Phk5 (342-367) have been synthesized and their interaction with calmodulin studied. In the case of Phk13 modified forms were also synthesized in which a tryptophan group was placed at position 4 or 21, as well as a form with tryptophan at position 4 and nitrotyrosine at position 21. From tryptic digestion, circular dichroism, and radiationless energy transfer measurements, it appears that Phk13 forms an elongated complex with calmodulin in which the peptide is in a non-helical conformation, probably bent into a hairpin-shaped structure, the connecting strand of calmodulin is extended and exposed to the action of proteolytic enzymes, and the peptide makes contact with both the N- and C-terminal half-molecules of calmodulin. In contrast, the Phk5 peptide has an alpha-helical conformation in the complex, which is relatively compact in shape.
Subject(s)
Calmodulin/chemistry , Peptide Fragments/chemistry , Phosphorylase Kinase/chemistry , Amino Acid Sequence , Animals , Calmodulin/analogs & derivatives , Calmodulin/isolation & purification , Calmodulin/metabolism , Cattle , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Fluorescence Polarization , Kinetics , Male , Molecular Sequence Data , Peptide Fragments/chemical synthesis , Peptide Fragments/metabolism , Phosphorylase Kinase/metabolism , Protein Conformation , Spectrometry, Fluorescence , Testis/metabolism , Trypsin , Tryptophan/analysisABSTRACT
Biphenyl metabolism in Aspergillus toxicarius occurs by successive hydroxylations in the 4- and 4'-positions, followed by conjugation with sulfate to produce 4-hydroxybiphenyl-O-sulfonic acid and 4,4'-dihydroxybiphenyl-O-sulfonic acid. The hydroxylation reactions normally occur only after a prolonged lag period after which the appearance of the monohydroxylated compound precedes the dihydroxylated compound. The accumulation of the monohydroxy compound is transient; therefore, it is an intermediate in the hydroxylating pathway. The onset of hydroxylating activity can be greatly accelerated when the culture is primed with the intermediate or product of the reaction (4-hydroxybiphenyl or 4,4'-dihydroxybiphenyl) at the time of biphenyl addition; a concentration of 0.05 mg 4,4'-dihydroxybiphenyl per ml produces optimal induction. Water-soluble conjugates of 4-hydroxybiphenyl and 4,4'-dihydroxybiphenyl were found in cultures of A. toxicarius grown in the presence of biphenyl plus inducer. The conjugate was shown to be the sulfate ester; no glucuronide or other conjugate species was found in any phase of the transformation. As with hydroxylating activity, the sulfotransferase activity appeared to be induced by the products of biphenyl metabolism.
