Fractionation, bovine brain electrophoresis

Fig. 1 Analysis by IFF In polyacrylamide gel (pH 3.5-10, 5% Amphollne, 3M urea) of the results of a fractionation of bovine brain homogenate with the Elphor VaP 21 operating In ZE mode. Sample supernatant of 300g of whole brain homogenized In 300 ml water, electrodlalyzed and centrifuged. Input at 3 ml/hr In the center channel (46). Buffer 10 mM sodium acetate, gH 5.0, 7 ml/ min. Electrophoresis conditions 218 V/cm, 85 mA, 6 C. The starting material Is In the left most lane (S). The fractions analyzed are shown In number. This Is a preliminary run and no optimization has been attempted. Hemoglobin and albumin from unremoved blood are Indicated.

The ADP-ribosylation reaction is stimulated by GTP, phospholipids, and various cellular factors, both membrane and soluble (2-9). Kahn and Gilman (7, 9) isolated a membrane protein termed ADP-ribosylation factor (ARF) that promoted the toxin-catalyzed ADP-ribosylation of Gso. The protein bound GTP in a reaction which was enhanced by NaCl and dimyristoyl phosphatidylcholine (9). It was proposed that ARF boimd directly to Gso (7) and Aat the ARF Gso complex served as the actual substrate in the toxin-catalyzed reaction (7). Tsai et cd. (10) demonstrated that ARF from bovine brain membranes activated the toxin directly rather than interacting with the substrate Gso. Other factors that enhanced the ability of choleragen to ADP-ribosylate Gso were identified in a soluble fraction from bovine brain. Two proteins that accoimted for most of the choleragen activation by the soluble fraction were resolved by ion exchange chromatography and separately purified. Each exhibited one major band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 

S-D-2-Acetamido-2-deoxyhexosidases have been purified from bovine brain tissue by electrophoresis, ion-exchange chromatography, and gel filtration. Two of the four fractions obtained exhibited both j8-D-2-acetamido-2-deoxy-galactosidase and -glucosidase activities, whereas the others each contained only one of these activities. 

Fig. 1. SDS-polyacrylamide gel electrophoresis of lAP substrates. Electrophoretic gels (12.5% polyacrylamide) were stained with Coomassie blue. Lane 1 Mj markers lanes 2-6 fractions obtained at purification steps of rat brain according to the procedure used for rabbit liver (see Table 1 in [31]) (2 cholate extract 3 DEAE-Sephacel 4 AcA-34 5 heptylamine-Sepharose 6 DEAE-Sephacel II) lane 1 the first peak of Fig. 2A lane 8 the second peak of Fig. 2A lane 9. rabbit liver Nj lane 10 bovine retinal transducin. Mj X 10" is shown by arrows...

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