Abstract
Two surrogate substrates, methyl 1-heptylthioacetothioate (HEPTAT) and methyl 1-hexylthioacetothioate (HEXTAT) were utilized to compare a new spectrophotometric assay with the standard radiochemical partition assay used to quantify juvenile hormone esterase (JHE) activity. The surrogate substrates were made with one common factor being a thiol ester moiety substituting for the ester moiety found in juvenile hormones (JHs) and a thioether replacing the 2,3-olefin of the JHs. As a result, nucleophilic attack by the serine residue of JHE at the carbonyl functional group results in a hydrolytic reaction and release of methanethiol. In the presence of Ellman's Reagent (DTNB) methanethiol will cleave the disulfide bond of DTNB resulting in a chromophore detectable at 405 nm. Methyl 1-hexylthioacetothioate and its oxygen ester analogue, methyl-l-hexylthioacetate, were compared for JHE activity. Statistical analysis of the slopes indicated a very small but significant difference between the hydrolytic rates for the thiol ester and oxygen ester. However, the data indicate that thiol esters can replace oxygen esters to quantify hydrolytic activity by the JHEs examined. Results gathered from different preparations of JHE including tissue culture media from a baculovirus expression system, affinity- and DEAE-purified enzyme, as well as insect hemolymph indicate an excellent correlation between the two assays. Isoelectric focusing of pure and crude JHE preparations resulted in coinciding peaks of hydrolytic activity when using the standard partition assay and the spectrophotometric assay, with no other peaks of activity found in the crude preparations with either substrate. Several esterase bands were found at different isoelectric points when gels were stained with a-naphthyl acetate. Substrates were further characterized by monitoring specific activity of JHE collected from hemolymph of larvae of Manduca sexta and Heliothis virescens.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 119-126 |
| Number of pages | 8 |
| Journal | Insect Biochemistry and Molecular Biology |
| Volume | 25 |
| Issue number | 1 |
| DOIs | |
| State | Published - 1995 |
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Keywords
- Colorimetric or spectrophotometric assay
- Juvenile hormone esterase
- Partition assay
- Surrogate substrates
ASJC Scopus subject areas
- Insect Science
- Biochemistry
- Molecular Biology
Cite this
Characterization of a spectrophotometric assay for juvenile hormone esterase. / McCutchen, Bill F.; Szekacs, Andras; Huang, Tien L.; Shiotsuki, Takahiro; Hammock, Bruce D.
In: Insect Biochemistry and Molecular Biology, Vol. 25, No. 1, 1995, p. 119-126.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Characterization of a spectrophotometric assay for juvenile hormone esterase
AU - McCutchen, Bill F.
AU - Szekacs, Andras
AU - Huang, Tien L.
AU - Shiotsuki, Takahiro
AU - Hammock, Bruce D.
PY - 1995
Y1 - 1995
N2 - Two surrogate substrates, methyl 1-heptylthioacetothioate (HEPTAT) and methyl 1-hexylthioacetothioate (HEXTAT) were utilized to compare a new spectrophotometric assay with the standard radiochemical partition assay used to quantify juvenile hormone esterase (JHE) activity. The surrogate substrates were made with one common factor being a thiol ester moiety substituting for the ester moiety found in juvenile hormones (JHs) and a thioether replacing the 2,3-olefin of the JHs. As a result, nucleophilic attack by the serine residue of JHE at the carbonyl functional group results in a hydrolytic reaction and release of methanethiol. In the presence of Ellman's Reagent (DTNB) methanethiol will cleave the disulfide bond of DTNB resulting in a chromophore detectable at 405 nm. Methyl 1-hexylthioacetothioate and its oxygen ester analogue, methyl-l-hexylthioacetate, were compared for JHE activity. Statistical analysis of the slopes indicated a very small but significant difference between the hydrolytic rates for the thiol ester and oxygen ester. However, the data indicate that thiol esters can replace oxygen esters to quantify hydrolytic activity by the JHEs examined. Results gathered from different preparations of JHE including tissue culture media from a baculovirus expression system, affinity- and DEAE-purified enzyme, as well as insect hemolymph indicate an excellent correlation between the two assays. Isoelectric focusing of pure and crude JHE preparations resulted in coinciding peaks of hydrolytic activity when using the standard partition assay and the spectrophotometric assay, with no other peaks of activity found in the crude preparations with either substrate. Several esterase bands were found at different isoelectric points when gels were stained with a-naphthyl acetate. Substrates were further characterized by monitoring specific activity of JHE collected from hemolymph of larvae of Manduca sexta and Heliothis virescens.
AB - Two surrogate substrates, methyl 1-heptylthioacetothioate (HEPTAT) and methyl 1-hexylthioacetothioate (HEXTAT) were utilized to compare a new spectrophotometric assay with the standard radiochemical partition assay used to quantify juvenile hormone esterase (JHE) activity. The surrogate substrates were made with one common factor being a thiol ester moiety substituting for the ester moiety found in juvenile hormones (JHs) and a thioether replacing the 2,3-olefin of the JHs. As a result, nucleophilic attack by the serine residue of JHE at the carbonyl functional group results in a hydrolytic reaction and release of methanethiol. In the presence of Ellman's Reagent (DTNB) methanethiol will cleave the disulfide bond of DTNB resulting in a chromophore detectable at 405 nm. Methyl 1-hexylthioacetothioate and its oxygen ester analogue, methyl-l-hexylthioacetate, were compared for JHE activity. Statistical analysis of the slopes indicated a very small but significant difference between the hydrolytic rates for the thiol ester and oxygen ester. However, the data indicate that thiol esters can replace oxygen esters to quantify hydrolytic activity by the JHEs examined. Results gathered from different preparations of JHE including tissue culture media from a baculovirus expression system, affinity- and DEAE-purified enzyme, as well as insect hemolymph indicate an excellent correlation between the two assays. Isoelectric focusing of pure and crude JHE preparations resulted in coinciding peaks of hydrolytic activity when using the standard partition assay and the spectrophotometric assay, with no other peaks of activity found in the crude preparations with either substrate. Several esterase bands were found at different isoelectric points when gels were stained with a-naphthyl acetate. Substrates were further characterized by monitoring specific activity of JHE collected from hemolymph of larvae of Manduca sexta and Heliothis virescens.
KW - Colorimetric or spectrophotometric assay
KW - Juvenile hormone esterase
KW - Partition assay
KW - Surrogate substrates
UR - http://www.scopus.com/inward/record.url?scp=0029167448&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0029167448&partnerID=8YFLogxK
U2 - 10.1016/0965-1748(94)00036-H
DO - 10.1016/0965-1748(94)00036-H
M3 - Article
VL - 25
SP - 119
EP - 126
JO - Insect Biochemistry and Molecular Biology
JF - Insect Biochemistry and Molecular Biology
SN - 0965-1748
IS - 1
ER -