Trypsin 0.25 percent

Tryptic activity measured in vitro with bovine trypsin. Tryptic activity and larval growth reported as percent of control where control equals 100%. Soybean inhibitor was Kunitz inhibitor. Percent relative growth determined by weight gain of S. exigua for 11 days on artificial diet containing the specified phytochemicals. 

Figure 2. Peptide maps (A-C) and MALDI-TOF mass spectra (D-F) of PVDF-bound transferrin (53 pmol) digested with trypsin in the presence of 50 pi of 1% RTX-100/10% acetonitrile/100 mM Tris, pH 8.0 (A,D), 1% octylglucopyranoside/10% acetonitrile/100 mM Tris, pH 8.0 (B,E), and 1% decylglucopyranoside/10% acetonitrile/100 mM Tris, pH 8.0 (CJF) as described in Materials and Methods. Ninety percent of the digestion was analyzed by HPLC ( 29 pmol based on Table I) and 0.5% ( 150 fmol) was used for MALDI-TOF mass spectrometry. Peptides 1 and 2 in A-C were amino terminally sequenced (Table II) and analyzed by MALDI-TOF mass spectrometry (Figure 3).

Figure 1 Relationships of S with interfacial tension and emulsifying activity of proteins. I, bovine serum albumin 2, /3-lactoglobulin 3. trypsin 4, ovalbumin 5, conalbuntin 6, lysozyme 7, K-casein 8, 9, I0, II, and 12, denatured ovalbumin by heating at 85°C for l, 2, 3, 4, and 5 min respectively 13, 14, 15, 16. 17, and 18. denatured lysozyme by heating at 85"C for l, 2, 3, 4, 5, and 6 min respectively 19, 20, 21, 22, and 23, ovalbumin bound with 0.2, 0.3, 1.7, 5.7, and 7.9 mol of sodium dodecyl sulfate/mol of protein respectively 24, 25, 26, 27, and 28, ovalbumin bound with 0.3, 0.9, 3.1,4.8, and 8.2 mol of linoleate/mol of protein respectively. Interfacial tension measured at corn oil/0.20c protein interface with a Fisher Surface Tensiontat Model 21. Emulsifying activity index calculated from the absorbance at 500 nm of the supernatant after centrifuging blended mixtures of 2 ml of corn oil and 6 ml of 0.5% protein in 0.01 M phosphate buffer, pH 7.4 S initial slope of fluorescence intensity (FI) vs. percent protein plot. 10 /al of 3.6 mM m-parinaric acid solution was added to 2 ml of 0.002 to 0.1% protein in 0.01 M phosphate buffer, pH 7.4, containing 0.002% SDS. FI was measured at 420 nm by exciting at 325 nm. (From Ref. 2. Reprinted by permission.)...

GM oilseed rape plants containing a gene encoding the protease inhibitor OCI, under the control of the CaMV 35S promoter, had measurable quantities of this transgene product in their leaves (0.2-0.4 percent of total soluble protein) but not in their pollen [16]. This finding was confirmed by Jouanin et al. [17], who also noted that Bowman-Birk soybean trypsin inhibitor (BBI) could not be detected in the nectar or pollen of GM oilseed rape plants which had measurable expression levels in leaves (gene also on the CaMV 35S promoter). 

Effects of trypsin (lOpg/mL) on CPT-I activity time course were similar to those of Nagarse, but there was no corresponding loss of malonyl-CoA inhibition even when the enzyme activity had been destroyed by about 60%, Figure 4. This data is in contrast to a recent report by Fraser et suggesting that trypsin at a concentration of lOpg/mL has no effect on CPT-I activity. Their data is presented as percent activity as a function of exposure time to trypsin with no indication of the specific activity of the enzyme before... 

To test this suggestion, trypsin was inhibited with the highly specific reagent Na carbobenzyloxylysylchloromethylketone (RCOCH2CI), labeled in the ketone carbon with 90 percent... 

Toxicity of 3,3 and 6,10 polyionenes to normal WI-38 cells and SV40 WI-38 cells in monolayer cultures was studied by the standard 3-day growth response. Thirty ml Falcon plastic culture flasks were seeded with 0.25 x 10 cells/flask in 5 ml of Eagle s medium -t-10% fetal calf serum. After 24 hours, the medium was replaced with the medium containing different concentrations (0-10 /ig/ml of 3,3 or 6,10 ionene polymer and the cells were allowed to grow for an additional three days. On the fourth day the cells were trypsinized and the number of cells per ml was calculated with the use of a Coulter particle counter. Three replicate flask cultures were analyzed at each concentration and the experiments were repeated twice. The growth factor (GF) for each treatment was calculated as the ratio of the number of cells/ml on day 4 to the number of cells/ml on day 1 (on the day of initiation of the treatments) and was expressed as percent of the control. The toxicity was plotted as a function of concentration as shown in Figures 3 and 4. 

Trypsin was processed from aqueous solution (25 mg/ml in 10 mM HCl) at a flow rate of 0.03 ml/min and contacted with 1.2 ml/min ethanol and 5.0 ml/min CO2. Reported percent activities are relative to the as-received commercial protein. 

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