Comparison of the Lowry and the Bradford protein assays as applied for protein estimation of membrane-containing fractions.

Although the Bradford protein estimation assay has found wide distribution, it has a number of drawbacks, which in some cases have been shown to produce erroneous results upon comparison to other, more precise, methods for protein estimation. It was found that the underestimation of the protein content of membrane-containing fractions cannot be overcome by pretreatment with NaOH or the detergents employed (Triton X-100, sodium dodecyl sulfate, 3[(3-cholamidopropyl)-dimethylammonio]propanesulfonic acid) and the protein estimates obtained do not agree with estimates obtained by the Lowry assay. Upon storage of fractions at -20 degrees C there is a considerable loss of dye binding activity, varying in accordance with the membrane content of the fractions, reaching up to 58% in the case of membrane-enriched fractions stored at -20 degrees C for 15 days. Pretreatment with the employed agents brought about an equal increase of dye binding capacity, specific for the individual fractions; however, none of these agents could recover the dye binding activity lost during several days of storage at -20 degrees C. It is suggested that the straightforward Bradford procedure has a rather limited scope of application, particularly concerning membrane-containing samples, and requires preliminary studies to determine its applicability according to the nature of the biological material examined.

Effects of vanadate on the assembly and disassembly of purified tubulin.

Sodium-orthovanadate (100-700 microM) added to purified pig brain microtubule protein (molar ratios 13-90 moles vanadate/mole tubulin) inhibits to a considerable extent the assembly (up to 65%) and the disassembly rates (up to 60%) of microtubules, as determined by turbidimetry. Vanadate added to preformed microtubules did not appreciably alter the turbidity level of the samples, however, the disassembly rates were decreased in the same manner as when vanadate was added prior to polymerization. Microtubule protein kept on ice for 3-6 hours became more susceptible to vanadate than freshly prepared protein. The effect of vanadate was independent of the GTP concentration at which the polymerization assays were performed (0.025 to 1 mM GTP). In the presence of taxol, which increases the rate and extent of microtubule formation, vanadate had no effect on assembly rates. Disassembly was inhibited, however, much less than in the presence of vanadate alone. Electron microscopy and polyacrylamide gel electrophoresis did not reveal differences between microtubules prepared in the presence or in the absence of vanadate. This is consistent with the notion that vanadate does not interfere with the interaction between tubulin and the high-molecular weight microtubule-associated proteins. Apparently vanadate brings about an allosteric change of the microtubule protein(s) resulting in the abnormal polymerization kinetics of tubulin found in our study. The above results may be relevant for studies where the effects of vanadate on intracellular motility are interpreted as being solely due to a specific inhibition of ATPases.