DNA quantitation with a benchtop fluorometer.
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Bisbenzimidazole↗
Biomedical subjects
S R Gallagher
Publications and source records attributed to S R Gallagher.
Plant polypeptides reversibly glycosylated by UDP-glucose. Possible components of Golgi beta-glucan synthase in pea cells.
In pea membranes, UDP[14C]Glc glycosylates a approximately 40-kDa polypeptide doublet. This label rapidly disappears if excess unlabeled UDP-Glc, or UDP, is added, indicating that the glycosylation is reversible, and suggesting that the glycosylated polypeptides might be intermediates in a glycosyl transfer reaction. Glycosylation of the doublet requires a divalent cation, the effective ions being the same (except for Zn2+) as those that activate Golgi-localized beta-glucan synthase (GS-I) activity. Treatments that inhibit GS-I also inhibit doublet glycosylation. The doublet is associated with Golgi (and to a minor extent with plasma) membranes and occurs also in the soluble fraction. The Golgi-bound doublet may be a component of the GS-I system. Immunological, inactivation, and fractionation evidence indicates that at least one other polypeptide is required in GS-I activity.
Cell Fractionation↗
Phenol-acetic acid-urea polyacrylamide gel electrophoresis of membrane proteins.
A phenol-acetic acid-urea polyacrylamide gel electrophoretic system (PAU-PAGE) was simplified by adaptation to a slab gel format, allowing the simultaneous comparison of up to 12 samples. The system fractionated most proteins according to molecular mass, although chemical reduction was required since certain proteins (e.g., bovine serum albumin) showed reduction-dependent shifts in mobility. Sodium dodecyl sulfate-PAGE of partially purified membrane proteins can be adversely affected by protein aggregation and proteolysis. PAU-PAGE, which solubilized aqueous insoluble proteins and rapidly inactivated proteases, was useful for assessing the polypeptide composition of plasma membrane preparations.
Acetates↗
Electrophoretic characterization of a detergent-treated plasma membrane fraction from corn roots.
Experiments were conducted to determine conditions essential for electrophoretic characterization of a detergent-extracted plasma membrane fraction from corn (Zea mays L.) roots. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) initially gave poor resolution of polypeptides in the plasma membrane fraction and, upon detergent treatment for purification of the proton-pumping adenosine triphosphatase (ATPase), showed no enrichment for a 100 kilodalton catalytic subunit characteristic of the ATPase. In contrast to SDS-PAGE, phenol urea acetic acid (PAU)-PAGE clearly resolved two polypeptides in the 100 kilodalton region that were enriched during detergent treatment and indicated at least one polypeptide forms a phosphorylated intermediate characteristic of the ATPase. Problems with SDS-PAGE were found to be caused, in part, by a combination of endogenous proteases and heat-induced aggregation of high molecular weight proteins. The usually standard procedure of boiling the sample prior to SDS-PAGE caused the aggregation of the 100 kilodalton polypeptides. By controlling for proteases using chymostatin and/or phenylmethane sulfonyl floride, and not boiling the sample prior to electrophoresis, two polypeptides were clearly resolved by SDS-PAGE in the 100 kilodalton region of Triton X-114-extracted membranes from corn, oat, barley, and tomato.
Journal Article↗
A sensitive diffusion plate assay for screening inhibitors of protease activity in plant cell fractions.
Proteolytic activity was detected, using a sensitive radial diffusion plate assay, in the plasma membrane fractions of corn (Zea mays L.) roots and from roots of several other plant species. The proteases could be effectively inhibited in corn with phenylmethane sulfonyl fluoride or chymostatin. Protease activity of oat roots, however, was not significantly reduced by these inhibitors. The results of diffusion plate assay were confirmed with the less sensitive azocasein assay using crude cell homogenates. Chymostatin and phenylmethane sulfonyl fluoride were effective in preventing protease degradation of polypeptides as revealed by electrophoresis. The diffusion plate assay uses a permanent support for a 0.75 millimeter thick agarose slab containing 200 micrograms per milliliter casein. By staining the fixed and dried gel with Coomassie blue R-250, proteolytic activity was visualized as a cleared area around the sample well with a detection limit of about 0.3 nanograms trypsin. The diffusion plate assay should prove useful for screening inhibitors of proteases where limited amounts of material are available, such as with plant cell fractions or highly purified proteins.
Journal Article↗
Effect of vanadate, molybdate, and azide on membrane-associated ATPase and soluble phosphatase activities of corn roots.
The effects of vanadate, molybdate, and azide on ATP phosphohydrolase (ATPase) and acid phosphatase activities of plasma membrane, mitochondrial, and soluble supernatant fractions from corn (Zea mays L. WF9 x MO17) roots were investigated. Azide (0.1-10 millimolar) was a selective inhibitor of pH 9.0-ATPase activity of the mitochondrial fraction, while molybdate (0.01-1.0 millimolar) was a relatively selective inhibitor of acid phosphatase activity in the supernatant fraction. The pH 6.4-ATPase activity of the plasma membrane fraction was inhibited by vanadate (10-500 micromolar), but vanadate, at similar concentrations, also inhibited acid phosphatase activity. This result was confirmed for oat (Avena sativa L.) root and coleoptile tissues. While vanadate does not appear to be a selective inhibitor, it can be used in combination with molybdate and azide to distinguish the plasma membrane ATPase from mitochondrial ATPase or supernatant acid phosphatase.Vanadate appeared to be a noncompetitive inhibitor of the plasma membrane ATPase, and its effectiveness was increased by K(+). K(+)-stimulated ATPase activity was inhibited by 50% at about 21 micromolar vanadate. The rate of K(+) transport in excised corn root segments was inhibited by 66% by 500 micromolar vanadate.
Journal Article↗