The prophenoloxidase from the wax moth Galleria mellonella: purification and characterization of the proenzyme

Kopáček, Petr; Weise, Christoph; Götz, Peter

doi:10.1016/0965-1748(95)00040-2

Cited by 91 publications

(58 citation statements)

References 27 publications

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“…Obtained values from diluted samples were multiplied with a dilution factor to calculate the phenoloxidase activity in 1 ml hemolymph. Phenoloxidase activity was expressed as change of absorbance in 1 ml hemolymph [19] (Fig. 1 C).…”

Section: Methodsmentioning

confidence: 99%

“…Phenoloxidase activity in hemolymph samples obtained from untreated larvae was measured according to the method described by Kopacek et al [19] using DL-3,4-dihydroxyphenylalanine (DOPA) as the substrate (4 mg/ml dissolved in distilled water). Hemolymph samples were collected, diluted 1:10 with ice-cold (10 mM sodium cacodylate, pH 6.5, in 20 mM CaCl 2 ) cacodylate buffer and stirred intensively (hemolymph pool 1).…”

Section: Methodsmentioning

confidence: 99%

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Purification and characterization of an inducible metalloprotease inhibitor from the hemolymph of greater wax moth larvae, Galleria mellonella

Wedde

Weise

Kopáček

et al. 1998

European Journal of Biochemistry

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In this paper, we report the detection, purification and characterization of the first metalloprotease inhibitor (IMPI) from invertebrates. IMPI was purified from the hemolymph of last-instar larvae of Galleria mellonella by precipitation with trichloroacetic acid and heat followed by affinity chromatography on a thermolysin-Sepharose column and gel filtration or reverse-phase high-performance liquid chromatography. For the detection of inhibitor activity, a new azocoll assay was established. IMPI was only detectable in larvae that had been injected with bacterial or fungal provocators, suggesting that it is induced nonspecifically during the humoral immune response. Injection of larvae with IMPI rendered them resistant to thermolysin, in quantities that normally would be lethal for them. IMPI was shown to be specific for metalloproteases. The molecular mass of IMPI was determined by mass spectrometry to be 8360 Da. Purified IMPI was heterogeneous, owing to different degrees of glycosylation with hexose/hexosamine and deoxyhexose residues. Ten cysteine residues were found in the molecule, and these are presumed to form five disulfide bridges. The amino terminus was blocked, but a partial amino-acid sequence starting from the thermolysin cleavage site was determined; this sequence exhibited no similarity with other known proteins, suggesting that the IMPI represents a new type of protease inhibitor. Keywords : metalloproteases; metalloprotease inhibitor ; insect immunity ; Galleria mellonella.Insects are particularly resistant to micro-organisms. The endogenous defense of insects is based on cellular and humoral immune responses. The latter includes the synthesis of a broad spectrum of potent antimicrobial proteins that act directly against invading micro-organisms [1,2]. The response of insects to wounding or infection includes hemolymph coagulation and an activation of the prophenoloxidase cascade. The latter process is activated by serine proteases and regulated by serine-protease inhibitors in order to avoid excessive activation of and damage to host tissues [3,4]. In addition, protease inhibitors are thought to inhibit undesired proteolytic enzymes released by damaged cells or invading pathogens. Entomopathogenic fungi are the only group of micro-organisms that can infect insect hosts directly through their chitinous exoskeleton. Fungal extracellular proteases contribute to both integument penetration and digestion of host proteins. They are reported to determine the host specificity and the virulence of the producing fungal species [5,6]. Protease inhibitors detected in the cuticle or hemolymph of several insect species have been proposed to regulate not only endogenous proteases but also act against toxic fungal proteases hibitors and one cysteine protease inhibitor, but no metalloprotease inhibitor [12].Recently, it was reported that injection of zymosan or heatinactivated yeast cells resulted in reduced or delayed mortality of G. mellonella larvae after injection of living pathogenic fungal cells [13]. T...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Purification and characterization of an inducible metalloprotease inhibitor from the hemolymph of greater wax moth larvae, Galleria mellonella

Wedde

Weise

Kopáček

et al. 1998

European Journal of Biochemistry

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“…To assess PO and proPO activity we followed a modified protocol from Kopáçek et al (1995). From each individual, the haemolymph concentrations of active PO and the proenzyme proPO were measured.…”

Section: (D) Lysozyme Assaymentioning

confidence: 99%

Juvenile immune system activation induces a costly upregulation of adult immunity in field cricketsGryllus campestris

et al. 2004

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Inducible immune defence may allow organisms a state-dependent upregulation of costly immunity in order to minimize the risk of anticipated future parasitism. The basic costs of elevated immune activity might involve a reduction in other fitness-related traits as well as an increased risk of immunopathology. In male field crickets Gryllus campestris we experimentally investigated the condition-dependent effects of immune system activation in nymphs on immunity and physiological condition during adulthood. Following a nymphal injection of bacterial lipopolysaccharides, adult males showed significantly elevated levels of two major immune parameters, i.e. haemolymph antibacterial activity and the concentration of prophenoloxidase (proPO). By contrast, the active enzyme, phenoloxidase (PO), did not increase, suggesting a strategic longterm upregulation of the inactive proenzyme proPO only. This may help avoid the cytotoxic effects associated with high standing levels of the active enzyme. The nymphal immune insult further caused a reduction in adult haemolymph protein load, suggesting a long-term decline in overall metabolic condition. Nymphal food availability positively affected adult lysozyme activity, while PO and proPO concentrations were not affected. Our data thus suggest the long-term upregulation of immunity in response to antigenic cues as an adaptive, yet costly, invertebrate strategy to improve resistance to future parasitism.

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“…Native forms of these enzymes are usually characterised by having molecular masses >100 kDa (Barrett, 1987b;Hall et al, 1995;Kopácek et al, 1995). Such studies have assayed substrate specificity of purified or crude extracts of PO spectrophotometrically.…”

Section: Introductionmentioning

confidence: 99%

“…Purified proPO and cuticle PO have been obtained as a single protein or as a dimer (Andersson et al, 1989;Aspán & Söderhäll, 1991;Hall et al, 1995;Kopácek et al, 1995).…”

Section: Introductionmentioning

confidence: 99%

Cresolase, catecholase and laccase activities in haemocytes of the red swamp crayfish

Cárdenas

Dankert

2000

Fish & Shellfish Immunology

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Phenoloxidase activity in crayfish haemocyte lysates and extracts of haemocyte membranes were studied using native PAGE and SDS-PAGE gels and staining for cresolase, catecholase and laccase activities. The activation of the proenzyme, prophenoloxidase to phenoloxidase, in native PAGE was demonstrated following exposure to SDS. By staining samples separated in SDS-PAGE followed by renaturation, a high molecular mass phenoloxidase activity was identified in both the soluble and membrane fractions of haemocyte preparations. The membrane-associated activity appeared at only relatively high molecular mass (>300 kDa), and could easily be eluted from membranes using detergents or NaCl. Further, this membrane-associated activity has a catecholase activity but not the cresolase activity seen in the soluble preparations. In addition, several other phenoloxidase enzymes were identified with di#erent relative mobilities (250, 80, 72 and 10 kDa). Crayfish haemocytes also contained laccase activity, thought to be restricted to cuticle sclerotisation in the integument. Laccase activity in haemocytes might aid in the formation of capsule used to contain pathogens. Academic Press

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The prophenoloxidase from the wax moth Galleria mellonella: purification and characterization of the proenzyme

Cited by 91 publications

References 27 publications

Purification and characterization of an inducible metalloprotease inhibitor from the hemolymph of greater wax moth larvae, Galleria mellonella

Purification and characterization of an inducible metalloprotease inhibitor from the hemolymph of greater wax moth larvae, Galleria mellonella

Juvenile immune system activation induces a costly upregulation of adult immunity in field cricketsGryllus campestris

Cresolase, catecholase and laccase activities in haemocytes of the red swamp crayfish

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