Oyster glycogen

Soluble proteins were extracted from developing barley grains at 13–15 DAA using 2 volumes (3 volumes for BE) of grinding solution [50 mM imidazole–HCl (pH 7.4), 8 mM MgCl₂, 500 mM 2-mercaptoethanol, 0.1% (w/v) acarbose, 12.5% (v/v) glycerol] relative to grain fresh weight. Developing grains (10–15 DAA) of rice (Oryza sativa subspecies japonica “Nipponbare”) grown in a paddy field of Akita Prefectural University during the summer months under natural condition was used as control. SS activity staining was performed using a gel containing 0.8% (w/v) oyster glycogen (Sigma-Aldrich, G8751) as primer (Miura et al. 2018 (link)). BE activity staining was assessed using a gel containing 0.0001% oyster glycogen (Yamanouchi and Nakamura 1992 (link)). DBE activity staining was performed using gels containing 0.4% (w/v) potato amylopectin (Sigma-Aldrich, A8515) as described in Fujita et al. (1999 (link)). PUL activity staining was performed using a gel including 1.34% (w/v) red pullulan (Megazyme) as described in Fujita et al. (2009 (link)).

Stock solutions of buffered glycogen at either pH 4, 5, 6 or 7 were made by mixing 5 ml of 250 mM HEPES (Fisher Scientific, city state) or 5 ml of 100 mM lactic acid, with 20 ml of 10 mg/ml glycogen (oyster glycogen, Sigma Chemical Co), and 20 ml of phenol-red-free RPMI 1640 medium as a source of divalent cations (Lonza, Walkersville, MD). The pH of each stock solution was then adjusted with HCl and sterile filtered.
Genital fluid, saliva (diluted 1:1000 in saline) or saline as a negative control (5 μl) were mixed with the glycogen stock solution (45 μl) in tubes and incubated at 37°C for 120 minutes. After incubation, 10 μl was transferred to wells of a microtiter plate and the pH in the wells adjusted to 7 by addition of 43 μl of 250 mM HEPES (pH 7) and 30 μl of phenol-red-free RPMI-1640 medium.
Glycogen was detected by color development using a modification of a previously-described assay [13 (link)] which consisted of addition to each well of 50 μl acetic acid (1.7 M), 50 μl potassium iodate (0.1 N) and 50 μl of potassium iodide (0.1 N). Plates were agitated and after 15 min read at 565 nm. The amount of glycogen in wells was calculated based on a standard curve made from dilutions of glycogen.

Approximately equal weights of pancreas per sample were used for purification. Tissues were homogenized in PBS using a TissueLyser II (Qiagen), centrifuged at 13,000 x g at 4 °C for 10 min, and the crude lysate was collected. Ethanol was added to a final concentration of 40%, centrifuged at 10,000 x g for 10 min at 4 °C, and the supernatant was collected. Amylase was precipitated by addition of 1 mg of oyster glycogen (Sigma-Aldrich) according to [32 (link)] followed by shaking on ice for 5 min. It was then pelleted by centrifugation at 5000 g for 3 min at 4 °C. The samples were washed, re-suspended in PBS, and glycogen digested by incubation at 30 °C for 20 min [33 (link)]. Samples were stored at − 80 °C in aliquots to avoid repeated freeze/thaw cycles. Protein concentration was determined using Thermo Scientific’s Coomassie Plus™ (Bradford) Assay kit according to the manufacturer’s instructions. For native PAGE gels Amylase extracts were separated on 7.5% Mini-PROTEAN® TGX™ gels (Bio-Rad), but in their native form (no boiling, no SDS). The gel was then placed in 1% pre-warmed starch solution and incubated for 1 h at 37 °C (without shaking). Subsequently it was transferred to Lugols solution for approx. 1 min and then straight to PBS. This was followed by two further brief washes in PBS and imaging using a white light transilluminator.