Biomedical subjects

P K Parrack

Publications and source records attributed to P K Parrack.

7 recordsLinked to original sources

PubMed1995

Aging of tubulin monomers using 5,5'-bis(8-anilino-1-naphthalenesulfonate) as a probe.

The fluorescent probe bis(8-anilino-1-naphthalenesulfonate) (bis-ANS) has been used to monitor the time- and temperature-dependent aging of tubulin, whereby new hydrophobic binding sites of lower affinity are generated on the protein [Prasad, A. R. S., et al. (1986a) Biochemistry 25, 739-742]. We carried out a detailed analysis of this phenomenon and found that, in addition to antimitotic drugs like colchicine or vinblastine, other parameters, viz., low temperature and protein stabilizers (e.g., glycerol and sucrose), inhibit the extent of enhanced binding of bis-ANS. Moreover, the generation of additional bis-ANS binding sites are also suppressed at high concentrations of tubulin. Cleavage of the carboxy-termini of tubulin (bound to bis-ANS) by subtilisin causes a significant reduction in the enhanced fluorescence, but has no effect on the high-affinity binding site of bis-ANS. All of these observations can be explained by the correlation of the presence of additional binding with the dissociation of heterodimeric tubulin into monomers. Enhanced binding of bis-ANS is due to tubulin dimers that have undergone dissociation, resulting in a loosening of its tertiary structure with the generation of a plethora of hydrophobic sites.

Anilino Naphthalenesulfonates↗

PubMed1992

Bis-ANS as a specific inhibitor for microtubule-associated protein induced assembly of tubulin.

5,5'-Bis[8-(phenylamino)-1-naphthalenesulfonate] (bis-ANS), the fluorescent probe which binds to tubulin, inhibits its assembly into microtubules [Horowitz et al. (1984) J. Biol. Chem. 259, 14647-14650]. The results described in this paper demonstrate that bis-ANS is quite distinct from other well-known microtubule inhibitors in its specificity of action. The inhibitory potentials of bis-ANS and its three structural analogues ANS, Prodan [6-propionyl-2-(dimethylamino)naphthalene], and NSA (naphthalenesulfonic acid) have been compared. It is found that they can be arranged in the following order according to their polymerization inhibitory potentials: bis-ANS approximately equal to Prodan much greater than ANS greater than NSA. Interestingly, the naphthalene nucleus is sufficient to cause inhibition of polymerization. Detailed experiments were carried out to examine the mode of assembly inhibition by aminonaphthalenes at the molecular level, using bis-ANS as a representative. It was found that there was little or no effect of bis-ANS on the assembly of tubulin when polymerization was induced by assembly promoters like taxol, DMSO, or glutamate, or on the assembly of subtilisin-digested protein (tubulin S), for all of which half-maximal inhibition could not be achieved even at 120 microM bis-ANS. On the contrary, bis-ANS acts as an inhibitor in the case of MAP- (MAP2 and tau) and poly(L-lysine)-induced assembly of tubulin, with half-maximal inhibitory concentrations ranging from 1.5 to 7.6 microM. Our results place bis-ANS as a novel inhibitor, which seems to specifically inhibit C-termini-mediated assembly. Of all assembly inhibitors known so far, none exhibits such selection.(ABSTRACT TRUNCATED AT 250 WORDS)

Anilino Naphthalenesulfonates↗

PubMed1992

Interaction of Prodan with tubulin. A fluorescence spectroscopic study.

The compound 6-propionyl-2-(N,N-dimethyl)-aminonaphthalene (Prodan), an efficient fluorescent probe for proteins, is shown to bind to tubulin. Detailed experiments on fluorescence enhancement, anisotropy and energy transfer were carried out to unravel the nature of Prodan-tubulin interaction and the Prodan-binding site on tubulin. It was found that Prodan binds to tubulin at a rigid site, with a stoichiometry of 1:1 and a dissociation constant of 20 microM. Competition experiments using 1,8-[3H]anilinonaphthalenesulfonate ([3H]ANS) indicated that the three aminonaphthalenes, ANS, bis(1,8-anilinonaphthalenesulfonate) and Prodan probably bind to a common region on the tubulin molecule.

2-Naphthylamine↗

PubMed1991

Methanog: a specialized database on methanogenic bacteria.

A specialized, interdisciplinary database on various types of related information on methanogenic bacteria is described. Derived from other sequence databases etc., this database collects information from many sources, including unpublished work from research laboratories working in this field, and makes them accessible from a single source, to interested scientists, free of cost. It is presently held in eight 48 T.P.I. floppy disks and can be run on any IBM PC under DOS 3.0 or above, making this database of particular interest to researchers with limited resources and on-line search/access facilities.

Amino Acid Sequence↗

PubMed1990

The carboxy terminus of the alpha subunit of tubulin regulates its interaction with colchicine.

Controlled proteolysis of goat brain tubulin by subtilisin was carried out to investigate regulatory aspects of the binding of colchicine to tubulin. Tubulin S, obtained by the cleavage of the carboxyl termini of both the alpha- and beta-subunits of tubulin by subtilisin, exhibited the following differences compared to native tubulin: (a) Reaction with colchicine, which has an optimum pH of 6.8, becomes independent of pH (in the range 5.7-8.0). (b) The colchicine-binding site, which is labile at 37 degrees C (t1/2 = 4-5 h), becomes highly stable (t1/2 greater than 12 h). (c) The affinity for colchicine is lowered. (d) This lowering of affinity arises from a faster dissociation (higher off rate) of the complex. The above characteristics of tubulin S were not shown by a partially digested hybrid in which the C-terminus of the beta-subunit alone was cleaved. The hybrid behaved very much like the undigested native protein. These results strongly suggest that the regulatory switch for colchicine-tubulin interaction is located in a small region (about 15 residues) of the C-terminus of the alpha-subunit of tubulin. Possibilities of the C-termini being involved in nonbonded contacts with the main body of tubulin are also noticed from the change in conformation between tubulin and tubulin S.

Animals↗

PubMed1984

Fibre diffraction of lithium DNA shows structural variability and deviation from a regular helical structure for the B-form.

Recently, reports have appeared which show structural variations in B-DNA and indicate deviations from a uniform helical structure. We report for the first time that these indications are also present in the B-form fibre diffraction patterns for the lithium salt of natural DNA. We have used an improved method of controlling the salt concentration in the fibres. Our results are based on the appearance and disappearance of meridional reflections on different layer lines depending upon the salt.

Animals↗

PubMed1984

A detailed study of Li-DNA fibres at various salt concentrations reveals a non-helical B-DNA and a possible similarity of solution and solid state structures.

A thorough investigation of salt concentration dependence of lithium DNA fibres is made using X-ray diffraction. While for low salt the C-form pattern is obtained, crystalline B-type diffraction patterns result on increasing the salt concentration. The salt content in the gel (from which fibres are drawn) is estimated by equilibrium dialysis using the Donnan equilibrium principle. The salt range giving the best crystalline B pattern is determined. It is found that in this range meridional reflections occur on the fourth and sixth layer lines. In addition, the tenth layer meridian is absent at a particular salt concentration. These results strongly suggest the presence of non-helical features in the DNA molecule. Preliminary analysis of the diffraction patterns indicates a structural variability within the B-form itself. Further, the possibility of the structural parameters of DNA being similar in solid state and in solution is discussed.

DNA↗