Pattern Of Proteolytic Activity Research Articles

Cloning and Characterization of a Ribonuclease, a Cysteine Proteinase, and an Aspartic Proteinase from Pitchers of the Carnivorous Plant Nepenthes ventricosa Blanco

Nepenthes ventricosa is a carnivorous plant with modified leaves, pitchers, that secrete hydrolases to digest trapped insects, thus obtaining supplemental nutrients. Using reverse transcriptase PCR (RT‐PCR), three cDNAs encoding one putative ribonuclease (NvRN1) and two putative proteinases (NvAP1, NvCP1) were cloned from N. ventricosa pitcher tissues. The deduced amino acid sequences of NvRN1, NvAP1, and NvCP1 are homologous to the S‐like RNase PD2 (Prunus dulcis), the aspartic proteinase NaAP3 (Nepenthes alata), and the cysteine proteinase CysP2 (Glycine max), respectively. NvRN1 and NvAP1 were expressed in pitcher tissues and male flowers. NvRN1 expression was also evident in female flowers. SDS‐PAGE activity gels showed that pitcher fluid samples contained at least six proteinases of differing molecular masses (124, 65, 61, 49, 36, and 33 kD) and a single 21‐kD RNase. Fluid samples from single pitchers showed no change in pattern of proteolytic activity over time, but samples from different pitchers exhibited differing activity patterns, indicating that separate pitchers may secrete proteinases of different molecular masses. Optimal proteinase activity at pH 3 and inhibition by treatment with pepstatin was consistent with previous studies showing that Nepenthes secrete aspartic proteinases. Proteinase activity was also inhibited by E‐64, indicating that cysteine proteinases could be present in pitcher fluid.

Extracellular protease activity of different Pseudomonas strains: dependence of proteolytic activity on culture conditions

This study investigated the effect of growth conditions on proteolytic activity of a Pseudomonas strain, named Pseudomonas sp. LBSA1, isolated from bulk raw milk. It was compared with three Pseudomonas chlororaphis and one Pseudomonas fluorescens strain from culture collections. Bacteriae were grown in a minimal salt medium. For all the strains, addition of 1% (v/v) skim milk to the growth medium was sufficient to induce protease production in 48-h culture. Addition of 1 mmol l(-1) calcium chloride permitted the detection of proteolytic activity of four strains in 48-h cultures but not for Pseudomonas sp. LBSA1. The five strains presented two patterns of proteolytic activity when grown in the minimal salt medium supplemented with 2% (v/v) skim milk at various temperatures for 48 h. Two electrophoretic protease patterns were also obtained from the zymogram of extracellular medium for the five strains. The growth conditions permitting protease production are variable and do not depend on the genus of the producing strain. For the first time a study on proteolytic activity of P. chlororaphis strains is reported. Among the tested criteria, zymograms of extracellular medium were the only ones that permitted distinguishing the P. chlororaphis strains from the P. fluorescens strain.

Levels of Ammonia, Nitrite, and Nitrate in Alfalfa Root Nodules Supplied With Nitrate

Protein content, protease activity, and levels of NH4+, NO2- and NO3- were determined in the plant and bacteroid fractions of alfalfa (Medicago sativa L. cv. Aragon) nodules supplied with NO3- (0—200mM). Protein of the host cell cytosol represented approximately 70 % of the total nodule protein. The effect of NO3- on plant cytosolic protein closely paralleled that on bacteroid protein. Protein of both nodule fractions remained constant upon exposure to NO3- concentrations below 20 mM and was reduced by 30% above 20 mM NO3-. Patterns of proteolytic activity were inversely correlated to those of the corresponding protein for both bacteroids and cytosol. The nodule level of NH4+ was basically unaffected by increasing added NO3- whereas NO2- accumulated in both bacteroids and plant cytosol, although to a greater extent in the latter. Likewise, NO3- accumulated in the cytosol with much higher relative values than those of NO2-. Only trace or null amounts of NO3- were found in the corresponding bacteroids. Linear correlations either between bacteroidal and cytosolic NO2-, or between NO2- in both fractions and cytosolic NO3- levels were highly significant. The overall results indicate that the whole nodule capacity of NO2- reduction is not sufficient to avoid accumulation of NO2-, and support the view that NO2- plays a more important role than NH4+ or NO3- itself as inhibitor of nodule activity.

Haemolysin and enzyme patterns of coagulase-positive staphylococci isolated from toxic epidermal necrolysis, Ritter's disease and impetigo contagiosa.

Summary Strains of Staphylococcus aureus isolated from toxic epidermal necrolysis and Ritter’s disease (named collectively “Ritter’s type” of TEN) and from extensive cases of impetigo contagiosa, in which the diagnosis of Ritter’s type of TEN was considered seriously, were assigned to group E (extensive lesions) and were compared with strains of S. aureus isolated from unequivocal impetigo contagiosa (group L—localised lesions); 24 of the 26 isolates examined belonged to phage-group II. Strains from groups E and L were generally similar in their patterns of proteolytic activity, low incidence of positive egg-yolk reaction, possession of lipase and production of hyaluronidase. The difference lay in the many isolates in group E that produced a low-titre lysin active against rabbit red cells that was not α-haemolysin. The average titre of lysin active against rabbit erythrocytes in group E was 355 MHD per mi and in group L 1008 MHD per mi. Reasons are given for supposing that this low titre rabbit cell lysin is identical with staphylococcal δ-toxin. It is suggested that δ-toxin may play a part in the pathogenesis of Ritter’s type of TEN and extensive impetigo.