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R A Keates

Publications and source records attributed to R A Keates.

At least 19 recordsLinked to original sources
PubMed1990

Traces of brain microtubule-associated proteins affect dynamic properties of microtubules.

A method is described for measuring the quantities of stable and dynamic microtubules in a population in vitro. The method exploits the tendency of dynamic microtubules to depolymerize rapidly after being sheared. Stable microtubules, such as those protected by microtubule-associated proteins (MAPs), are broken to a smaller size by shearing, but do not depolymerize into subunits. The usual difficulty with this procedure is that the tubulin released from the dynamic microtubules rapidly repolymerizes before the end point of depolymerization can be measured. This has been overcome by including a small quantity of tubulin-colchicine complex in the mixture to block the repolymerization. For a total of 24 microM tubulin in a polymerization mixture, 10 microM of the sample polymerized originally under the conditions used. When 1.05 microM tubulin-colchicine complex was added at the time of shearing, the dynamic microtubules depolymerized, but the tubulin was released was unable to repolymerize and a small fraction of stable microtubules that resisted shear-induced depolymerization could then be detected. When traces of MAPs (0.23-2.8% by mass) were included in the tubulin mixture, the fraction of stable microtubules increased from 5% in the absence of added MAPs to 41% in the presence of 2.8% MAPs. All the MAPs in the mixture were found in the stable fraction and this stable fraction forms early during microtubule assembly. Calculations on the extent of enrichment of MAPs in the stable fraction indicated that as little as 4% MAPs in a microtubule protected it from shear-induced disassembly. The results suggest that low levels of MAPs may distribute nonrandomly in the microtubule population.

Animals↗
PubMed1990

Synapsin-1 is found in a microtubule-associated complex of proteins isolated from bovine brain.

We have prepared microtubules from brain tissue by stabilizing the cellular microtubules in 6.7 M glycerol buffer, instead of the usual procedure which extracts the solubilized protein and then reassembles microtubules in vitro at some later time. There are substantial differences in the microtubule associated proteins obtained by the two methods, and brain spectrin is a major component of the stabilized microtubules. We have now modified the buffer used for the isolation of stabilized microtubules to minimize their tendency to aggregate. When the stabilized microtubules were further purified by sucrose density gradient centrifugation, we were able to distinguish previously unidentified polypeptides at 49, 74 (doublet), and 100 kilodaltons (doublet). These bands maintained staining intensity in the same proportion to tubulin as in the original homogenate, whereas background proteins were diminished in staining intensity. We now report the identification of the 74-kilodalton doublet polypeptides as synapsin-1 by peptide mapping. Synapsin-1 is a protein known to bind to brain spectrin and also to microtubules, and may thus serve as a linker between these cytoskeletal components.

Amino Acid Sequence↗
PubMed1988

Dynamic instability of sheared microtubules observed by quasi-elastic light scattering.

The kinetics of microtubule reassembly was studied in vitro by quasi-elastic light scattering (QELS). When microtubules assembled in the absence of microtubule-associated proteins (MAPs) were sheared, they rapidly depolymerized, recovered, and reassembled. The mean length of the recovered microtubules was the same as that observed just before shearing, implying that on average one fragment per original microtubule survived the fragmentation and recovery. When microtubules that contained 25 percent brain MAP were sheared, the fragments did not depolymerize extensively and the average length of the fragments decreased by a factor of 3 relative to the unsheared sample. The results support the dynamic instability model, which predicts that cellular microtubules are latently unstable structures protected on their ends by stabilizing caps.

Animals↗
PubMed1985

Association of fodrin with brain microtubules.

We have compared the polypeptide composition of microtubules isolated from bovine brain by the conventional in vitro reassembly method with those obtained by direct isolation of brain microtubules into a stabilizing buffer. The stabilizing buffer included 6.7 M glycerol to limit the rate of subunit exchange between assembled and unassembled states. The microtubule-associated proteins normally found by in vitro reassembly are also found in the stabilized preparation, but in smaller proportions. Fodrin, a brain membrane-associated protein believed to be homologous to spectrin, was found to be the most abundant component after tubulin in the stabilized microtubules. The ratio of tubulin to fodrin, 16:1 by mass, was almost constant at each stage of the preparation. Some actin was initially present in the stabilized microtubules, but was gradually lost during purification. When stabilized microtubules were diluted into cold aqueous buffer, they depolymerized and the recovered microtubule protein could then be purified by in vitro reassembly. The composition after this treatment resembled that of microtubules prepared initially by reassembly in vitro. The missing fodrin was found to be removed in the preliminary centrifugation and was unavailable for incorporation into growing microtubules during the in vitro assembly step. This suggests that the standard in vitro reassembly procedure for purification of microtubules may distort the composition of microtubule-associated proteins.

Animals↗
PubMed1984

Microtubule associated proteins in microtubule preparations made with and without glycerol.

Preparation of microtubule protein in the presence or absence of glycerol results in differences in polymerization properties and content of microtubule associated proteins. The variation in properties appears to result from the reduced proportion of microtubule associated proteins in preparations made with glycerol. I have used the colchicine binding assay to monitor recovery of active tubulin and have found that a single factor can account for the difference. During the in vitro assembly of microtubules from the crude brain homogenate, glycerol promotes polymerization of the bulk of the tubulin, while less than half is incorporated into microtubules in the absence of glycerol. Assembly of partly purified microtubule protein is not enhanced by glycerol however. Microtubule associated proteins present in the crude homogenate are almost completely incorporated into the microtubules regardless of the presence of glycerol, and their high content in glycerol-free preparations appears to be the trivial result of low tubulin recovery. The high affinity of microtubule associated proteins for the assembled microtubules has other consequences for in vitro studies of microtubule assembly, and critical concentration plots to determine the polymerization equilibrium constant can be distorted unless the preparation used has a high content of microtubule associated proteins.

Animals↗
PubMed1984

Inhibition of microtubule polymerization by micromolar concentrations of mercury (II).

Polymerization of microtubule protein in vitro has been tested in the presence of Hg2+. Inhibition occurs in the presence of Hg2+ concentrations lower than the molarity of the tubulin, even in the presence of 0.5 mM ethylenebis(oxyethylenenitrilo)tetraacetic acid. The estimated concentration of free Hg2+ under these conditions is less than 10(-20) M. The extent of inhibition is approximately stoichiometric in that the molar quantity of tubulin that fails to polymerize is comparable to or slightly greater than the total Hg2+ present as chelate. The affinity of the susceptible site for Hg2+ in microtubule protein must be exceptionally high and may not be protected by natural chelating agents in the cell. Preformed microtubules are rapidly depolymerized on addition of Hg2+ chelate. In vitro, inhibition is both prevented and rapidly reversed by 2-mercaptoethanol.

Animals↗
PubMed1981

Structural and functional alterations in microtubule protein from Chinese hamster ovary cell mutants.

We have examined mutant lines of Chinese hamster ovary cells that have increased resistance to the antimicrotubule drug Colcemid. Analysis of the functional properties of purified microtubule protein indicates that increased tolerance to the drug in vivo is reflected in altered properties of microtubules and tubulin in vitro. In this study, we have examined one series of related mutants and have found different microtubule alterations associated with each selection step. These changes include decreased Colcemid-binding affinity, an altered electrophoretic pattern of tubulin subcomponents, increased resistance to Colcemid inhibition of polymerization in vitro and, in one case, a decreased critical concentration for microtubule assembly. Characterized mutants of the class described here will be useful for probing the regulation of microtubule assembly in vivo.

Animals↗
PubMed1981

Stabilization of microtubule protein in glycerol solutions.

The consequences of short-term storage of microtubule protein at 0 degrees C have been examined and the ability of glycerol to prevent loss of activity has been evaluated. Three forms of activity monitored include colchicine-binding activity, critical concentration, and relative polymerizing activity. Colchicine-binding activity decayed continuously and glycerol increased the half-life roughly in proportion to its concentration (t 1/2 = 57 days in 4 M glycerol). Critical concentration remained relatively constant for 2-3 days and then increased rapidly in the absence of glycerol. This rapid increase was delayed in glycerol concentrations above 1 M. Relative polymerizing activity was based in this work on the slope of the critical concentration plot. In most cases, loss of activity was apparent after as little as 24 h. This was the first observed alteration in polymerization properties of the microtubule protein and glycerol concentrations below 3 M had little effect in slowing the decay. Degradation of microtubule-associated proteins may account for these changes in polymerization properties that occurred more rapidly than tubulin denaturation. Evidence for degradation was demonstrated by sodium dodecyl sulfate gel electrophoresis. Higher glycerol concentrations slowed this degradation.

Animals↗
PubMed1981

Inhibition of microtubule polymerization by the tubulin-colchicine complex: inhibition of spontaneous assembly.

The inhibition of microtubule polymerization by colchicine requires the formation of tubulin-colchicine complexes, and inhibition of polymerization is proportional to the concentration of tubulin-colchicine complexes rather than to the total concentration of colchicine. Because the formation of such complexes is slow relative to polymerization, the kinetics of complex formation obscure the kinetics of inhibition of polymerization. We have taken defined quantities of preformed tubulin-colchicine complexes, relying on their slow dissociation, and added these to microtubule protein, which was allowed to polymerize by temperature shift to 37 degrees C. The degree of polymerization was then determined by measurement of turbidity at 400 nm. An appropriate kinetic analysis allowed us to distinguish effects of inhibitor on initiation and elongation phase of polymerization, without resorting to the use of initiation inhibitors. The results are consistent with a reversible association of tubulin-colchicine complex with microtubule ends blocking further elongation (K1 = 0.16 micro M). Steady-state measurements suggest that copolymerization of tubulin-colchicine complex is a minor factor under the conditions used. By contrast, little inhibition of initiation was observed, possibly because tubulin-colchicine complex competes with the tubulin dimer, but not with the larger oligomers required for the initiation process.

Animals↗
PubMed1974

Protein kinase activity associated with isolated ribosomes from peas and lemna.

Protein kinase (ATP:protein phosphokinase) has been found associated with ribosomes prepared from 5-day-old etiolated pea shoots and sterile cultures of Lemna minor. The enzyme catalyzes the phosphorylation of ribosomal proteins in vitro and may be involved in the formation of phosphoproteins in the ribosomes in vivo. Protein kinase sediments with ribosomes during sucrose density gradient centrifugation through a buffer containing 0.02 m KCl. Seventy per cent of the enzymic activity dissociates from the ribosomes in 0.3 m KCl, but the remaining protein kinase remains firmly bound in 0.7 m KCl. Cyclic AMP does not modify the activity of these protein kinases in vitro. Benzyladenine inhibits the protein kinase, but only at high concentrations of this cytokinin.

Journal Article↗