Fractionation, bovine brain

Chloroform-methanol extracts of Borrelia burgdorferi were used for the identification of lipids and other related components that could help in the diagnosis of Lyme disease [58]. The provitamin D fraction of skin lipids of rats was purified by PTLC and further analyzed by UV, HPLC, GLC, and GC-MS. MS results indicated that this fraction contained a small amount of cholesterol, lathosterol, and two other unknown sterols in addition to 7-dehydrocholesterol [12]. Two fluorescent lipids extracted from bovine brain white matter were isolated by two-step PTLC using silica gel G plates [59]. PTLC has been used for the separation of sterols, free fatty acids, triacylglycerols, and sterol esters in lipids extracted from the pathogenic fungus Fusarium culmorum [60]. 

Since interferons appear to be species-specific, we investigated whether the ganglioside fraction from mouse brain was more potent in inhibiting antiviral activity of mouse fibroblast interferon than that obtained from heterologous brain extracts. As seen in Figure 1 bovine brain gangliosides were almost as potent... 

Although the occurrence of analogs of PAF in mammalian cells was always considered a possibility, Mueller et al. (1984) first reported the presence of 1-0 long-chain acyl-2-acetyl-glycero-3-phosphocholine in rabbit neutrophils. Later, Tokumura et al. (1989) isolated a vasopressor active phosphatidylcholine fraction from bovine brain. Treatment of the fraction with phospholipase C yielded a diglyceride component which was converted to the t-butyldimethylsilyl derivative. Analysis of the latter by gas-liquid chroma-... 

It is currently not clear as to how many PTX substrates there are and, of these, how many may co-exist in any given tissue. Brain has been reported as containing three PTX substrates of Mr 39000, 40000 and 41000 [73]. In heart and fat two PTX substrates were reported [163-165], Human erythrocytes appear to have one PTX substrate of A/r 40000 [29] which can be fractionated into two [151]. Prolonged exposures of autoradiograms of human erythrocyte, bovine brain, human neutrophil and rat liver PTX substrates reveal still a third substrate of apparent Mt = 43 000 [166,167]. As illustrated in Fig. 7, even more, up to five distinct proteins, can be visualized if membrane proteins are fractionated by SDS-PAGE in the presence of an urea gradient [151]. The functional difference between these PTX-labeled bands is not known at present. 

Soft ionization techniques such as electrospray ionization and matrix assisted laser desorption are now routinely used to determine the mass of large hydrophilic polymers like proteins (27). However, as is usual for the ionization process, the presence of sails and detergents, which is common for biological samples, can affect the process significantly. The use of the on-line capillary reversed-phase HPLC in combination of the electrospray mass spectrometer (LC/MS) has made it possible to analyze such samples directly (10,16, 28). When GAP-43 isolated from the membrane fractions of bovine brain was analyzed, a single major peak with a minor peak corresponding to a phosphorylated species was observed (Fig. la). To study the posttranslational modifications in detail, the protein was digested with specific proteases such as lysyl... 

Fig. 1. Mass spectrometric analysis of GAP-43 purified from membrane fractions of bovine brain, (a) A deconvoluted mass spectrum of GAP-43. A dcconvoluted mass spectrum of the N-terminal peptide before reduction (b) and after reduction (c). Peaks formed by oxidation of Met were also observed.

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.

U.L Lane 2 Bovine brain homogenate soluble fraction,... 

Kontro P, Marnela K M, and Oja S S (1980) Free amino acids m the synaptosome and synaptic vesicle fractions of different bovine brain areas Bram Res 184, 129-141... 

Galactosylceramide can be conveniently isolated from bovine brain tissue. If this organ is extracted successively with acetone, diethyl ether and then hot aqueous ethanol, sphingolipids are obtained in the final fraction. These precipitate on cooling and the residue is washed with acetone and dissolved in hot acetic acid. On cooling they can again be precipi-... 

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. 

Bovine brain lipid extract (Folch Fraction I) was purchased from Sigma (St. Louis, MO). Cholesterol was purchased from Avanti Polar Lipids (Alabaster, AL). PtdIns(4,5)P2 was from Calbiochem (La Jolla, CA). All lipids were solubilized in chloroform at the concentration of 25 mg/ml, 20 mg/ml, and 1 mg/ml, respectively, and stored at —20°. 

Mitochondria are isolated from bovine brain or liver. The starting material should be fresh in order to obtain intact mitochondria. The tissues are purchased directly from a slaughterhouse, cut into smaller pieces on site, and transported to the lab immersed in ice-cold mitochondrial isolation buffer (see following). This is an adaptation of more generalized protocols for subcellular fractionation by differential centrifugation (Graham and Rickwood, 1997). 

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. 

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