Purification and characterization of hemolymph juvenile hormone esterase from the cricket, Gryllus assimilis

Abstract

Juvenile hormone esterase (JHE) from the serum of the cricket, Gryllus assimilis, was purified to homogeneity in a four‐step procedure involving polyethylene glycol precipitation, hydrophobic interaction FPLC, and ion exchange FPLC. This procedure could be completed in 4 days and resulted in a greater than 900‐fold purification with greater than 30% recovery. The purified enzyme exhibited a single band on a silver‐stained SDS PAGE gel and had an apparent subunit molecular mass of 52 kDa. The native subunit molecular mass, determined by gel permeation FPLC, was 98 kDa, indicating that JHE from Gryllus assimilis is a dimer of two identical or similar subunits. The turnover number of the purified enzyme (1.41 s–1), KM(JH‐III) (84 ± 12 nM) of nearly‐purified enzyme, and kcat/KM (1.67 × 107 s–1 M–1) were similar to values reported for other well‐established lepidopteran and dipteran JHEs. JHE from Gryllus assimilis was strongly inhibited by the JHE transition‐state analogue OTFP (octylthio‐1,1,1‐trifluoro‐2‐propanone; I50 = 10–7 M) and by DFP (diisopropyl fluorophosphate; I50 = 10–7 M). The shapes of the inhibition profiles suggest the existence of multiple binding sites for these inhibitors or multiple JHEs that differ in inhibition. Isoelectric focusing separated the purified protein into 4 isoforms with pIs ranging from 4.7–4.9. N‐terminal amino acid sequences (11–20 amino acids) of the isoforms differed from each other in 1–4 positions, suggesting that the isoforms are products of the same or similar genes. Homogeneously purified JHE hydrolyzed α‐napthyl esters, did not exhibit any detectable acetylcholinesterase, acid phosphatase, or aminopeptidase activity, and exhibited only very weak alkaline phosphatase activity. JHE exhibited a low (11 μM) KM for long‐chain α‐naphthyl esters, indicating that JHE may have physiological roles other than the hydrolysis of JH‐III. Purification of JHE represents a key step in our attempts to identify the molecular causes of genetically‐based variation in JHE activity in G. assimilis. This represents the first homogeneous purification of JHE from a hemimetabolous insect. Arch. Insect Biochem. Physiol. 49:41–55, 2002. © 2002 Wiley‐Liss, Inc.

Tissue and stage-specific juvenile hormone esterase (JHE) and epoxide hydrolase (JHEH) enzyme activities and Jhe transcript abundance in lines of the cricket Gryllus assimilis artificially selected for plasma JHE activity: Implications for JHE microevolution

“…Stock-specific rates of JHE secretion into the hemolymph have yet to be directly studied in any Gryllus species. Finally, multiple JHE isoforms in G. assimilis have been identified that differ in up to 4 residues of the 13-20 N-terminal amino-acid sequences of the mature proteins (Zera et al, 2002;Crone et al, 2007). This suggests the possible existence of multiple Jhe genes that encode multiple JHE enzymes in this species.…”

Jhe in Gryllus assimilis: Cloning, sequence-activity associations and phylogeny

“…JHE had been previously purifi ed to homogeneity and characterised from the high activity and control lines (Zera et al, 2002). In the current study, a cDNA library from G. assimilis was constructed from fat body and midgut tissues taken from a mixture of 50 mid-last stadium (days 4-7, post moult) individuals from high activity, low activity and unselected control lines.…”

“…The cDNA library was initially screened by PCR to verify that a Jhe cDNA was contained within it, and to obtain an amplicon that could be used for further library screening. Degenerate oligonucleotides (5′-CNGARGCNGCICCNGARGT-3′, 5′-GCNGCICKCATNCKYTTYTG-3′) (nomenclature as per Sambrook et al, 1989) that bracketed the ends of a 20 amino acid sequence of the N-terminus of one isoform (JHE-3) of the mature JHE protein (Table 1; Zera et al, 2002) were used to initially screen ~10 3 pfu (1 μl) of the unamplifi ed library. The PCR reactions were performed in 50 μl and contained the following fi nal concentrations: 1×PCR buffer, 1.5 mM MgCl 2 , 2 mM each dNTP, 0.25 μM each primer and 0.5 units Taq DNA polymerase (Invitrogen, Carlsbad, CA).…”

“…Four hemolymph JHE isoforms were purifi ed to homogeneity from the cricket Gryllus assimilis; these isoforms differed among themselves in up to four of 13-20 residues at the N-terminus of the mature protein (Table 1; Zera et al, 2002). We used these amino acid sequences to design a probe that was used in a hybridisation screen of a G. assimilis fat body and midgut cDNA library.…”

“…Table 1. N-terminal amino acid sequences of the isoforms of the mature JHE protein of G. assimilis reported in Zera et al (2002) Isoform Sequence JHE-1 (minor) AEAPD EVEKA xGx (K) JHE-2 AEAAP EVEVA QKRMR AAAV JHE-3 AEADP EVEVA QxRMR GAVVP JHE-4 (minor) AEAAP EVEVA QKRMR AAVVP (L) "x" refers to an unidentifi ed residue; letter in brackets indicates ambiguity in the identity of the amino acid at that site, which could be either the residue designated by the letter above the brackets or within the brackets; "minor" means that band intensity of the isoform was much less than the JHE-2 and JHE-3 isoforms on an IEF gel (see Figure 4 of Zera et al, 2002).…”