The alkaline proteases of Argiope and their possible role in web digestion

Abstract

The digestive fluid of Argiope was assayed for its proteolytic activity with selected natural and synthetic substrates.1. Peak proteolytic activity was observed a t pH 7.8. This was coincident with the pH of the digestive fluid.2. Elastolytic and weak chymotryptic activities were detected. Limited activity toward collagen was also observed. Activity toward substrates for trypsin, leucine aminopeptidase and carpboxypeptidase A and B was absent.3. Evidence demonstrating the in vitro digestion of web fibroin was obtained. The viscid spiral was susceptible to a variety of proteases; the radial fibers to the spider enzyme alone.4. Of a series of inhibitors studied, EDTA had the greatest effect upon the spider proteases. This effect was temperature dependent.There is a growing body of information on the digestive proteases of invertebrates. The introduction of synthetic substrates and inhibitors now allows additional criteria for comparing the phylogenetic relationship between these and the vertebrate proteases (Neurath et al., '67; Barnard, '73).To our knowledge the most recent study on the digestive enzymes of spiders is that of Pickford ('42). She observed considerable proteolytic activity in both tissue extracts and gut fluids at alkaline pH values. In the present study we have sought to identify the specific catalytic nature of these proteases.Breed et al. (' 64) and Peakall ('72) have presented evidence, based on radioactive tracer studies, that orb-weaving spiders are capable of digesting their own web. The present study examines the effect of the digestive fluids on the orb-web fibroin. MATERIALS AND METHODSMature female Argiope were collected locally and identified by the criteria of Kaston ('52) as A. aurentia. Each spider was held gently with a cotton swab while fluid expressed from its mouth parts was collected in a 2O-pl pipette. The fluid was stored at -20°C until studied. The extent to which venom contributed to the fluid so expressed has yet to be evaluated. J. EXP. ZOOL.. 202: 213-222. Thermal stabilitySpider digestive fluid was diluted to a protein concentration of 0.5 mg/ml in 0.05 M borate buffer, pH 7.8 and pre-equilibrated at a temperature selected between 15" to 70°C at 5" intervals. The dilute enzyme mixture was then maintained at the selected temperature for 20 minutes before being rapidly brought to 25°C for assay with casein as described below. Enzyme assaysGeneral protease activity was assayed by a modified method of Kunitz ('47). The reaction mixture consisted of 2.5 ml 1% Hammarsten casein (Sigma Chemical Co.) in 0.05 M borate-KC1-NaOH buffer, pH 7.8 and 0.3 ml of distilled water. After equilibration for ten minutes a t 37"C, the reaction was initiated by the addition of 0.2 ml of diluted enzyme and allowed to incubate for 30 minutes. The reaction was terminated by the addition of 4.0 mi 5% trichloroacetic acid (TCA) and set aside for one hour. The precipitate was removed by filtration and the absorbance of the supernatant at O.D.zso was measured. Blank values were obtained by add...

Moult-related changes in ampullate silk gland morphology and usage in the araneid spider Araneus cavaticus

“…The spiders used to make these determinations were collected in the field and reared in rectangular cages in the laboratory (Tillinghast & Kavanagh 1977) until they ecdysed. They were killed at various times after ecdysis ranging from immediately after the removal of the legs from the exuvium to 32.6 days.…”

Recombinant protein blends: silk beyond natural design

“…The expression of the artificial gene results in the production of the desired recombinant protein, which is ultimately extracted from the host and purified (Figure 1). Genetic engineering has been utilized to produce recombinant fibrous proteins [3][4][5][6][7], with spider silks as one area of focus due to the remarkable material properties (e.g., high tensile strength, elasticity) [8,9]. Collagen fibers are also an outstanding group of fibrous proteins, supporting physiological functions throughout the human body based on combinations of sequence chemistry, different combinations of chains and complex structural hierarchy to achieve both biological and mechanical properties [10].…”

Astacin family metallopeptidases and serine peptidase inhibitors in spider digestive fluid

“…The digestive peptidases of orb-weaving spiders are necessary not only for consumption of prey, but also for the spider's recycling of its old orb webs into new (Tillinghast and Kavanagh 1977;Kavanagh and Tillinghast 1983;Townley and Tillinghast 1988). For some orb-weavers, including the araneid Argiope aurantia Lucas 1833, peptidases also play a role in the emergence of young spiders from the egg sac (Foradori et al 2002).…”

“…The most comprehensive studies to date are those of Mommsen (1978aMommsen ( ,b,c,d, 1980. In earlier studies (Tillinghast and Kavanagh 1977;Kavanagh 1979;Kavanagh and Tillinghast 1983), two peptidases were isolated from the digestive fluid of A. aurantia (though additional peptidases were demonstrated and partially characterized). Designated Argiope protease B and Argiope protease D, they had estimated molecular masses of 17.6 and 20.2 kDa, respectively, slightly alkaline pH optima, were cationic at physiological pH (about 7.8), and were shown to be metallopeptidases.…”

“…Mebs found three peptidases in the venom of the theraphosid Vitalius roseus (Mello-Leitão 1923) (formerly Pamphobeteus roseus). By size-exclusion chromatography, all three had molecular masses of about 11 kDa (but see Discussion regarding peptidase molecular masses estimated by this method), and they, like the A. aurantia digestive peptidases (Tillinghast and Kavanagh 1977;Tugmon and Tillinghast 1995), had alkaline pH optima, did not cleave trypsin substrates, and were unaffected by serine peptidase inhibitors.…”