Synthesis of a Molt-Inhibiting Hormone of the American Crayfish Procambarus Clarkii, and Determination of the Location of Its Disulfide Linkages

Abstract

A molt-inhibiting hormone (Prc-MIH) of the American crayfish, Procambarus clarkii, a member of the type II CHH family, was chemically synthesized and the location of its three disulfide linkages was determined. Prc-MIH consists of 75 amino acid residues and was synthesized by a thioester method. Two peptide segments, Boc-[Cys(Acm)(7,24,27), Lys(Boc)(19)]-Prc-MIH(1-39)-SCH(2)CH(2)CO-Nle-NH(2) and H-[Cys(Acm)(40,44,53), Lys(Boc)(42,51,67)]-Prc-MIH(40-75)-NH(2), were prepared using peptides obtained via the Boc solid-phase method. Condensation of the building blocks in the presence of silver chloride, 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine, and N, N-diisopropylethylamine, followed by removal of the protecting groups, gave the reduced form of Prc-MIH(1-75)-NH(2). This product was converted to the native form of Prc-MIH (synthetic Prc-MIH) in a buffer which contained cysteine and cystine. The synthetic Prc-MIH showed the same behavior by RP-HPLC and biological activity assays as the natural Prc-MIH. The disulfide bond between Cys7 and Cys44 was determined by isolation of a fragment from an enzymatic digest of the synthetic Prc-MIH by RP-HPLC, followed by mass analysis. The disulfide bonds between Cys24 and Cys40 and between Cys27 and Cys53 were determined by comparing the elution position of an enzymatic digest of the synthetic Prc-MIH with authentic chemically synthesized samples, which contained three types of possible disulfide linkages.

Crustacean Peptide Hormones: Structure, Gene Expression and Function

“…The results showed that rMIH-B had three disulfide bonds connected between Cys 7 and Cys 44 , Cys 24 and Cys 40 , and Cys 27 and Cys 53 . This arrangement was identical with that of other CHHfamily peptides reported previously (Kegel et al 1989;Huberman et al 1993;Martin et al 1993;Yasuda et al 1994;Gasparini et al 1994;Aguilar et al 1995Aguilar et al , 1996Nagasawa et al 1999;Kawakami et al 2000).…”

“…10). This arrangement is identical to those of other CHH-family peptides determined thus far (Kegel et al 1989;Huberman et al 1993;Martin et al 1993;Yasuda et al 1994;Aguilar et al 1995Aguilar et al , 1996Nagasawa et al 1999;Kawakami et al 2000;Katayama et al 2001) and, therefore, it is likely that this arrangement of disulfide bonds is also the same as in the native Pej-SGP-I. These results indicate that the difference in conformation between rPej-SGP-I-OH and rPej-SGP-I-amide is caused not by the variation of disulfide bond arrangement, but by the conformational change of the C-terminal region.…”

“…The disulfide bond arrangement in the native MIH was also determined in the same manner, and the same results as those in the case of rMIH were obtained. This disulfide pairing is essentially the same as those in other CHH-family peptides whose disulfide bond arrangements were determined previously (Kegel et al 1989;Huberman et al 1993;Martin et al 1993;Yasuda et al 1994;Gasparini et al 1994;Aguilar et al 1995Aguilar et al , 1996Nagasawa et al 1999;Kawakami et al 2000), suggesting that the type II peptide share a similar conformation with those of type I CHH-family peptides.…”

Changes in the Amounts of the Molt-Inhibiting Hormone in Sinus Glands during the Molt Cycle of the American Crayfish, Procambarus clarkii

“…Subsequently, the beads were washed five times with blocking buffer (0.01 M phosphate buffer, pH 7.0, containing 0.1% casein, 0.4 M NaCl, 0.01 M MgCl 2 and 0.1% NaN 3 ). The primary antibody-coated beads were incubated with 150 µ l of standard synthetic Prc-MIH (Kawakami et al, 1999(Kawakami et al, , 2000Sonobe et al, 2001) solution or sample solution for 14 hr at 4°C. The beads were incubated with biotinylated secondary antibody, anti-Prc-MIH(55-75)-NH 2 IgG solution (100 ng/ml of blocking buffer), for 1.5 hr at 25°C with shaking (180 strokes/min), and then reacted with streptavidin-β-D-galactosidase conjugate (0.25 U/ml, Roche Diagnostics, Germany) for 1 hr at 25°C with shaking.…”

Chemical synthesis of the ubiquitinated form of histone H3 and its effect on DNA methyltransferase 1

“…[Lys (Ub) 18 ,Cys(Acm) 96,110 ]H3.1 (14) was partially purified by gel filtration (75%), after which, the Acm groups were removed. We first attempted the removal by silver acetate in an aqueous acetic acid solution, 32 but the removal of the Acm groups were not complete, even after a 24-hour reaction (data not shown). It was recently reported that a Pd catalyst could be used to efficiently remove Acm groups.…”