Isolation and characterization of juvenile hormone esterase from hemolymph of Lymantria dispar by affinity- and by anion-exchange chromatography

Nussbaumer, Christa; Hinton, Andrew; Schopf, Axel; Stradner, Andrea; Hammock, Bruce D.

doi:10.1016/s0965-1748(00)00002-3

Cited by 14 publications

(13 citation statements)

References 19 publications

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“…The stained gel was used as a reference to determine the position of the native proteins in the unstained gel from which the native proteins were excised. Isoelectric gels were stained with IEF stain and placed on a shaking incubator at 60 RPM for 45 min; subsequently, they were destained in a 40% methanol/10% acetic acid solution for 2-4 h with solution changes every 30 min, or with a 20% methanol/5% acetic acid solution overnight (St. Leger et al 1994;Nussbaumer et al 2000). The novel proteins of immunized individuals and proteins present in control individuals were excised from the unstained gel.…”

Section: Electroelutionmentioning

confidence: 99%

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Inducible immune proteins in the dampwood termite Zootermopsis angusticollis

Rosengaus

Cornelisse

Guschanski

et al. 2006

Naturwissenschaften

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Dampwood termites, Zootermopsis angusticollis (Isoptera: Termopsidae), mount an immune response to resist microbial infection. Here we report on results of a novel analysis that allowed us to electrophoretically assess changes in hemolymph proteins in the same individual before and after exposure to a pathogen. We demonstrate that contact with a sublethal concentration of the entomopathogenic fungus Metarhizium anisopliae (Deuteromycotina:Hypomycetes) induces the production of protective proteins in nymphs, pseudergates (false workers), and soldiers. Termites exposed to an immunizing dosage of fungal conidia consistently showed an enhancement of constitutive proteins (62-85 kDa) in the hemolymph as well as an induction of novel proteins (28-48 kDa) relative to preimmunization levels. No significant differences in protein banding patterns relative to baseline levels in control and naïve termites were observed. Incubating excised and eluted induced proteins produced by immunized pseudergates or immunized soldiers with conidia significantly reduced the germination of the fungus. The fungistatic effect of eluted proteins differed significantly among five colonies examined. Our results show that the upregulation of protective proteins in the hemolymph underscores the in vivo immune response we previously recorded in Z. angusticollis.

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Section: Electroelutionmentioning

confidence: 99%

“…Novel protein bands were identified as those that were consistently visible in immunized individuals but absent in controls. Gel pieces were electroeluted using a Model 422 Electro-Eluter (BioRad), and the eluted proteins were then stored at −80°C (Nussbaumer et al 2000).…”

Section: Electroelutionmentioning

confidence: 99%

Inducible immune proteins in the dampwood termite Zootermopsis angusticollis

Rosengaus

Cornelisse

Guschanski

et al. 2006

Naturwissenschaften

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“…Recently, Kanaya and Kobayashi (2000), isolated and characterized a protein from silkworm hemolymph, able to increase the activity of a recombinant protein (luciferase) by approximately 6,000 times. Nevertheless, few studies have succeeded in isolating and characterizing the factors involved in these effects (Nussbaumer et al 2000;Shishikura et al 1996Shishikura et al , 1997Moon et al 1995;Ochanda et al 1992). These factors, once identified and isolated, can be of great importance in the optimization of cell growth, viral replication or recombinant protein production, leading to more efficient cell cultivations and to final products at lower costs.…”

Section: Introductionmentioning

confidence: 99%

Enhancing effect of a protein from Lonomia obliqua hemolymph on recombinant protein production

et al. 2008

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Gene expression in animal cells allows large scale production of proteins used for either structure and function studies or therapeutic purposes. Maximizing recombinant protein production is necessary to optimize cell growth and protein expression. Some studies have demonstrated the presence of pharmacologically active substances in insect hemolymph. In this work, we have identified and purified a protein from Lonomia obliqua hemolymph able to increase the production of the rabies virus glycoprotein, expressed in Drosophila melanogaster S2 cells, by about 59%.

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“…JHE from several holometabolous insects has been purifi ed to homogeneity or near homogeneity using both affi nity chromatography employing JHE transition-state analogues (Abdel-Aal and Hammock, 1985;Hanzlik and Hammock, 1987;Venkatesh et al, 1990;Nussbaumer et al, 2000;Thomas et al, 2000) and classical techniques (Coudron et al, 1981;Rudnicka and Jones, 1987;Valaitis, 1991Valaitis, , 1992Vermunt et al, 1997;Campbell et al, 1998;Nussbaumer et al, 2000). Typically, the affi nity protocol has resulted in higher yields (> 50%; Venkatesh et al, 1990;Abdel-Aal and Hammock, 1986) than classical approaches (< 25% yield; Rudnicka and Jones, 1987;Valaitis, 1992;Vermunt et al, 1997;Campbell et al, 1998).…”

Section: Discussion Jhe Purifi Cationmentioning

confidence: 99%

“…(Thomas et al, 2000). b Molecular mass and amino-acid sequence data indicate that these two enzymes are not the same protein (Nussbaumer et al, 2000).…”

Section: Jhe Isoformsmentioning

confidence: 99%

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

Zera

Sanger

Hanes

et al. 2001

Arch Insect Biochem Physiol

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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.

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Isolation and characterization of juvenile hormone esterase from hemolymph of Lymantria dispar by affinity- and by anion-exchange chromatography

Cited by 14 publications

References 19 publications

Inducible immune proteins in the dampwood termite Zootermopsis angusticollis

Inducible immune proteins in the dampwood termite Zootermopsis angusticollis

Enhancing effect of a protein from Lonomia obliqua hemolymph on recombinant protein production

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

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