Assaying proteinases with azocoll

Chavira, Ricardo; Burnett, Thomas J; Hageman, James H.

doi:10.1016/0003-2697(84)90242-2

Cited by 328 publications

(162 citation statements)

References 12 publications

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“…Thermolysin and thermolysin-inhibiting activities were measured with the azocoll assay according to the method of Chavira et al [18]. The method was modified and adapted for measurements with a microtiterplate photometer.…”

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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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%

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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“…RESULTS Hydrolysis of the protein substrate azocoll by three related enzymes was determined as follows: the incubation mixture contained 5 mg azocoll (19493, Calbiochem, Frankfurt, FRG) and 1 sg of either TSP-1 or t-kallikrein or 25 ng of pancreatic trypsin in a total volume of 1.1 ml of 0.1 M TrisHCl, pH 8.5. After 1 h incubation at 37°C changes in absorbance at 520 nm were recorded in the clear filtrate, previously freed from the insoluble azocoll as described [22]. Blank values, obtained in the absence of enzyme were subtracted in each case.…”

Section: Assay For Proteolytic Activitymentioning

confidence: 99%

“…TSP-1, and both t-and p-kallikrein are 'highly specific for arginine amides and are restricted to few substrates whereas pancreatic trypsin has a much less restricted specificity. When tested on the protein substrate azocoll, an insoluble, collagen-dye conjugate [22], TSP-1 and t-kallikrein only showed marginal proteolytic activity whereas the same substrate was much more rapidly (> lOO-fold) hydrolysed by pancreatic trypsin (table 2). These results again emphasize the highly restricted proteolytic activities of TSP-1 as well as those of tand p-kallikrein [28,29].…”

Section: Comparison Of Substrate Specificity Of Tsp-imentioning

confidence: 99%

A secretable serine proteinase with highly restricted specificity from cytolytic T lymphocytes inactivates retrovirus‐associated reverse transcriptase

Simon

Fruth

Kramer³

et al. 1987

FEBS Letters

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TSP-1, a murine T cell specific proteinase, is expressed in cytolytic T lymphocytes and secreted upon their interaction with antigen bearing target cells. In searching for possible extracellular substrates of the enzyme in the physiological environment of cytolytic effector cells, we have investigated the proteolytic activity of TSP-1 on retroviral proteins. It is shown that reverse transcriptase derived from the retrovirus Moloney murine leukemia virus is inactivated by TSP-1 via limited proteolysis. The data suggest the possibility that cytolytic T lymphocytes are able to interfere with retroviral replication by secreting a serine proteinase which degrades viral proteins.

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“…Proteolytic activity of His 6 Amq was tested by incubation of the protein (0.6 M and 1.2 M) with a suspension of azocoll (1.5 mg) for 30 min at 30°C and subsequent spectrophotometric detection of soluble dye-coupled peptides in the supernatant at 520 nm (13).…”

Section: Methodsmentioning

confidence: 99%

N-Acetylanthranilate Amidase fromArthrobacter nitroguajacolicusRü61a, an α/β-Hydrolase-Fold Protein Active towards Aryl-Acylamides and -Esters, and Properties of Its Cysteine-Deficient Variant

et al. 2006

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N-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldine degradation by Arthrobacter nitroguajacolicus Rü61a. Sequence analysis and secondary structure predictions indicated that Amq is related to carboxylesterases and belongs to the ␣/␤-hydrolase-fold superfamily of enzymes; inactivation of (His 6 -tagged) Amq by phenylmethanesulfonyl fluoride and diethyl pyrocarbonate and replacement of conserved residues suggested a catalytic triad consisting of S155, E235, and H266. Amq is most active towards aryl-acetylamides and aryl-acetylesters. Remarkably, its preference for ring-substituted analogues was different for amides and esters. Among the esters tested, phenylacetate was hydrolyzed with highest catalytic efficiency (k cat /K m ‫؍‬ 208 mM ؊1 s ؊1 ), while among the aryl-acetylamides, o-carboxy-or o-nitrosubstituted analogues were preferred over p-substituted or unsubstituted compounds. Hydrolysis by His 6 Amq of primary amides, lactams, N-acetylated amino acids, azocoll, tributyrin, and the acylanilide and urethane pesticides propachlor, propham, carbaryl, and isocarb was not observed; propanil was hydrolyzed with 1% N-acetylanthranilate amidase activity. The catalytic properties of the cysteine-deficient variant His 6 AmqC22A/ C63A markedly differed from those of His 6 Amq. The replacements effected some changes in K m s of the enzyme and increased k cat s for most aryl-acetylesters and some aryl-acetylamides by factors of about three to eight while decreasing k cat for the formyl analogue N-formylanthranilate by several orders of magnitude. Circular dichroism studies indicated that the cysteine-to-alanine replacements resulted in significant change of the overall fold, especially an increase in ␣-helicity of the cysteine-deficient protein. The conformational changes may also affect the active site and may account for the observed changes in kinetic properties.

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Assaying proteinases with azocoll

Cited by 328 publications

References 12 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

A secretable serine proteinase with highly restricted specificity from cytolytic T lymphocytes inactivates retrovirus‐associated reverse transcriptase

N-Acetylanthranilate Amidase fromArthrobacter nitroguajacolicusRü61a, an α/β-Hydrolase-Fold Protein Active towards Aryl-Acylamides and -Esters, and Properties of Its Cysteine-Deficient Variant

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