Simultaneous Preparation of Both Enantiomers of Juvenile Hormones Labeled at C-10 with Tritium at High Specific Activity

Maxwell, Robert A.; Anderson, R. J.; Schooley, David A.

doi:10.1006/abio.2002.5660

Cited by 5 publications

(3 citation statements)

References 17 publications

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“…[10S-3 H(N)]-JH III diol (20 Ci/mmol) was from a batch previously synthesized (Maxwell et al, 2002a). The activity of rec-Bommo-JHDK was monitored using the partition assay (Maxwell et al, 2002b).…”

Section: Biochemical Characterization Of Rec-bommo-jhdkmentioning

confidence: 99%

Juvenile hormone diol kinase, a calcium-binding protein with kinase activity, from the silkworm, Bombyx mori

Schooley

2005

Insect Biochemistry and Molecular Biology

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Section: Biochemical Characterization Of Rec-bommo-jhdkmentioning

confidence: 99%

Juvenile hormone diol kinase, a calcium-binding protein with kinase activity, from the silkworm, Bombyx mori

Schooley

2005

Insect Biochemistry and Molecular Biology

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“…1) and II diol (compound 4), (10R)- [10-3 H]JH III diol (compound 5), (10S,11S)- [10-3 H]JH diol (compound 3), and racemic [10-3 H]JH ethyl ester diol (compound 7) were previously synthesized in this laboratory (17). Enantiomerically pure, unlabeled JH diol was prepared from racemic JH using methods described previously (17). The specific activity and purity of each JH diol were determined by mass spectrometry (Finnigan MAT SSQ 710) using electron impact ionization (70 eV).…”

Section: Synthesis Of Labeled Jh and Its Metabolitesmentioning

confidence: 99%

“…For this reason, detailed kinetic analyses were performed with these potential substrates. Each [10-3 H]JH diol was carefully synthesized and enantiomerically purified (17). Very little has been done to identify the naturally occurring enantiomer of JH diol.…”

Section: Fig 3 Rplc Purification Of Jhdk By Wax Chromatographymentioning

confidence: 99%

Juvenile Hormone Diol Kinase

Maxwell

Welch

Schooley

2002

Journal of Biological Chemistry

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Manduca sexta juvenile hormone diol kinase (JHDK) catalyzes the conversion of juvenile hormone (JH) diol to JH diol phosphate. JHDK may be the first example of a phosphotransferase directly involved in the catabolism and inactivation of a lipid-soluble hormone. JHDK is an enzyme crucial for secondary metabolism of JH and possesses high specificity and catalytic efficiency for JH diol. In this study, the purification and characterization of native JHDK are described; its enzymatic properties are examined; and its role in cellular JH metabolism is explored. Using a variety of potential substrates, we show that JHDK has a preference for ATP, but will catalyze the formation of JH diol phosphate with GTP as the phosphate donor. JHDK has a nanomolar K m for JH I diol and a low micromolar value for MgATP. JH II and III diols also serve as phosphate acceptors with low micromolar K m , whereas other diol derivatives of terpenoid esters structurally similar to JH metabolites are not phosphorylated. The reaction proceeds via a sequential Bi Bi mechanism. JHDK is active as a homodimer with a subunit molecular mass of 20 kDa. JHDK binds 5-p-fluorosulfonylbenzoyladenosine and is inhibited by micromolar levels of Ca 2؉ .Juvenile hormones (compound 1; see Fig. 7) are sesquiterpenoids produced in the corpora allata of insects. They travel to target tissues through the hemolymph via a juvenile hormone (JH) 1 -binding protein, where they serve to modulate development, metamorphosis, and sexual maturation (1). During metamorphosis and larval development, JH titers are strictly regulated through biosynthesis and catabolism (2, 3). JH esterase (JHE) and JH epoxide hydrolase are regarded as the enzymes responsible for JH catabolism. However, recent studies have identified a hormonal role for JH acid (compound 2; see Fig. 7) (4, 5), suggesting that JHE is more than an inactivating enzyme. Recently, a cytochrome P450 terpenoid hydroxylase was discovered in the corpora allata of Diploptera punctata; this enzyme may play a role in the temporal suppression of JH titer in the cockroach by metabolizing JH III to 12-hydroxy-JH III (6).Most identifications of JH metabolites in the past have involved studies using either hemolymph or whole organisms, making it difficult to distinguish the role of intracellular JH metabolism. Two studies have been conducted using Manduca sexta tissues dissected and maintained in vitro, one with Malpighian tubules (7) and the other with fat body and imaginal discs (8). The classic model for JH metabolism in insects consists of two primary pathways of inactivation: epoxide hydrolysis to form the diol metabolites and ester cleavage to form the acid metabolites. However, there have been many reports indicating very polar JH metabolites (9 -15). We identified and characterized JH diol phosphate (JHDP; compound 11; Fig. 1), one such polar metabolite, and suggested that it is the principal end product of JH I metabolism in M. sexta (11).We isolated and characterized JH diol kinase (JHDK) from the Malpighian tubules o...

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