Table 1.
Characteristics of gliadins from Triticum aestivum and human salivary basic proline-rich proteins.1
Table 2.
Comparison of amino acid sequences of salivary basic proline-rich protein 2 (PRB2) from human saliva and wheat omega-5 protein from Triticum aestivum.*
Figure 1.
Hydrolysis of gliadin-derived enzymatic substrates.
Dental plaque was suspended in saliva ion buffer to an OD620 of 1.2. Substrates Z-YPQ-pNA, Z-QQP-pNA, Z-PPF-pNA and Z-PFP-pNA were added to final concentrations of 200 µM. Substrate conversion was monitored spectrophotometrically at 405 nm. A, hydrolysis measured during the 0–6 hr time interval; B, hydrolysis measured after 6 h, 24 h and 48 h; C, substrates incubated for 48 h in saliva ion buffer only (control).
Figure 2.
Degradation of a mixture of gliadins (G) by oral plaque microorganisms.
Dental plaque was suspended in saliva ion buffer to an OD620 of 1.2. Gliadin (Sigma) was added to a final concentration of 250 µg/ml. After various incubation times at 37°C 100 µl aliquots were removed, boiled and analyzed by SDS PAGE. A, lane 1, Molecular weight standard; lane 2: plaque bacterial suspension only (t = 0); lane 3: plaque suspension + gliadin (t = 0); lane 4: 25 µg gliadin (t = 0); B, lanes 5 to 10: plaque suspension + gliadin, sampled after t = 0, t = 2 h, t = 4 h, t = 6 h, t = 24 h and t = 48 h, respectively. C, gliadin incubated for the same time intervals in saliva buffer only (control).
Figure 3.
Degradation of gliadin-derived peptides by dental plaque bacteria.
The 33-mer or the 26-mer were added to a final concentration of 250 µg/ml to a suspension of dental plaque bacteria in saliva ion buffer (OD620 1.2). After t = 0 h, 2 h and 5 h, 100 µl aliquots were removed, boiled and subjected to RP-HPLC. A and C, chromatograms of the 33-mer and 26-mer peptides incubated in dental plaque suspension; B and D, 33-mer and 26-mer peptides incubated in saliva ion buffer only (control).
Figure 4.
Degradation of the 33-mer in human whole saliva (WS).
The 33-mer was added to a final concentration of 250 µg/ml to WS collected from eight individual subjects. In each of the saliva samples, the degradation of the 33-mer was monitored. The average peak area and standard deviation of the 33-mer incubated for 0 h, 2 h, 5 h, 8 h and 24 h in the eight WS samples are shown. Control: 33-mer only, incubated in saliva ion buffer for the same time intervals.
Figure 5.
Determination of the pH activity profile of oral gliadin-degrading enzymes.
A, Gliadin zymography. Sonicated plaque bacterial supernatant was aliquoted into eight equal fractions. Each fraction containing 90 µg protein was loaded onto a gliadin zymogram gel. After electrophoresis and renaturing the gel was cut and individual lanes were developed in 20 mM Tris buffers exhibiting pH values from 3 to 10. Far left lane: molecular weight standard. B, Z-YPQ-hydrolysis. Plaque bacteria were suspended 20 mM Tris buffers varying in pH from 2 to 10 to a final OD620 of 1.2. Suspensions were mixed with Z-YPQ-pNA (200 µM). Substrate hydrolysis was monitored spectrophotometrically at 405 nm. A representative graph of 3 independent experiments is shown.
Figure 6.
Isoelectric focusing profile of the gliadin degrading enzymes.
Proteins in sonicated plaque bacteria supernatant were separated based on isoelectric point by liquid iso-electric focusing (IEF). The enzymatic activity in each of the 10 IEF fractions was established by gliadin zymography. Far left lane, MW standard; S1: plaque supernatant sample before IEF.