A new tubulin-binding protein

A new brain protein is described which forms an insoluble complex with tubulin, with concomitant stoichiometric hydrolysis of GTP. The complex contains a maximum of one tubulin-binding protein (MW 52,500) per two tubulin dimers. The tubulin-binding protein (TBP) does not compete with colchicine, but in the presence of microtubule-associated proteins tubulin appeared less accessible to it. Proteins such as TBP might sequester tubulin and thereby function either to inhibit indiscriminate polymerization, or to promote ordered nucleation by maintaining high local concentrations.     

Properties of SDS-polyacrylamide gels highly cross-linked with N,N'-diallyltartardiamide and the rapid isolation of macromolecules from the gel matrix.

Polyacrylamide gels cross-linked with N,N′-diallyltartardiamide (DATD) are, in contrast to gels cross-linked with N,N′-methylenebisacrylamide (Bis), readily solubilized by periodic acid (Anker, H. S., 1970, FEBS Lett.7, 293), thus permitting efficient analyses of electrophoretically separated, labeled biological material. The capacities of polyacrylamide gels, cross-linked with Bis and DATD, to serve as media for electrophoretic separation of proteins, were compared. As DATD-cross-linked gels were inferior to equimolar Bis-crosslinked gels with 5% cross-linking (C_Bis = 5%) by the criteria of more pronounced swelling, markedly softer gels and, less concentrated and bended protein zones on electrophoresis and subsequent staining, gels cross-linked with different percentage CDATD were examined. The water regain of DATD-cross-linked gels, the retardation coefficients, and free mobilities of different proteins in equimolar Bis- and DATD-cross-linked gels were determined. When the DATD concentration in gels was increased to C_DATD = 27%, gels assumed physical characteristics comparable to those cross-linked with Bis at C_Bis = 5%. We report further the rapid, facile isolation of protein bands out of the gel matrix cross-linked with DATD. However, the isolation procedure results in an irreversible loss of biological activity.     

The effects of deuterium oxide (2H2O) on the polymerization of tubulin in vitro

The initial rate and final extent of polymerization of both bovine brain tubulin and sea urchin egg tubulin were enhanced in the presence of ²H₂O. The yields were increased in association with the elevation of the ²H₂O concentration. ²H₂O also reduced the critical concentration for polymerization of brain tubulin. Thermodynamic analysis was attempted using the temperature dependence of the critical concentration for polymerization in the presence of ²H₂O. We obtained linear van 't Hoff plots and calculated thermodynamic parameters which were positive and were increased with the elevation of the ²H₂O concentration. The enhancement of the polymerization of tubulin by ²H₂O could, therefore, be the result of the strenghening of intra-and/or inter-molecular hydrophobic interactions of the tubulin molecules. We believe that the increase in lenghth and number of microtubules of the mitotic spindles in the dividing cells of the eukaryotes with ²H₂O may be caused by the direct involvement of ²H₂O in the polymerization of tubulin.     

The nucleotide sequence of the yeast ARG4 gene

The complete nucleotide sequence of a 2296-bp DNA fragment containing the yeast (Saccharomyces cerevisiae) ARG4 gene has been determined. This gene specifies the synthesis of the arginine biosynthetic enzyme, argininosuccinate lyase (EC 4.3.2.1). The sequence contains one major open reading frame of 463 codons, giving a calculated M_r of 52010 for the protein, in good agreement with the experimentally determined value of 53 000. The sequence upstream from the ARG4 gene shares structural features in common with other yeast genes subject to general amino acid control.     

Activation of pigeon erythrocyte adenylate cyclase by cholera toxin partial purification of an essential macromolecular factor from horse erythrocyte cytosol

A cytosolic, macromolecular factor required for the cholera toxin-dependent activation of pigeon erythrocyte adenylate cyclase and cholera toxin-dependent ADP-ribosylation of a membrane-bound 43 000 dalton polypeptide has been purified 1100-fold from horse erythrocyte cytosol using organic solvent precipitation and heat treatment. This factor, 13 000 daltons, does not absorb to anionic or cationic exchange resins, is sensitive to trypsin or 10% trichloroacetic acid and is not extractable by diethyl ether. Activation of adenylate cyclase by cholera toxin requires the simultaneous presence of ATP (including possible trace GTP), NAD⁺, dithiothreitol, cholera toxin, membranes and the cytosolic macromolecular factor. Reversal of cholera toxin activation of adenylate cyclase, and of the toxin-dependent ADP-ribosylation, requires the presence of the cytosolic factor. The ability of the purified cytosolic factor to influence the hormonal sensitivity of liver membrane adenylate cyclase may provide clues to its physiological functions.     

Cloning of genes for bacteriophage T5 stable RNAs

One EcoRI-generated fragment (440 basepairs) and two EcoRI/HindIII fragments (220 and 960 basepairs) from the deletion region of T5 phage have been inserted into the phage λ XIII and the plasmid pBR322 as vectors. Recombinant DNA molecules were studied by hybridization with in vivo ³²P-labeled T5 4–5 S RNAs on nitrocellulose filters. Two-dimensional polyacrylamide gel electrophoretic fractionation and fingerprint analysis of the RNAs eluted from the filters were carried out to identify RNAs coded by cloned fragments. For the accurate localization of the genes for these RNAs, RNA-DNA hybrids were treated with T1 and pancreatic RNAases, and the eluted RNA fragments stable against RNAase action were electrophoresed. It was shown that the EcoRI¹440 fragment contains the gene for tRNA 10 (tRNA^Asp), the EcoRI/HindIII¹220 fragment contains the gene for RNA III (107 bases) and parts of the genes for RNA I (107 bases) and tRNA 12 (tRNA^His), and the EcoRI/HindIII¹960 fragment contains only a part of the gene for tRNA 9 (tRNA^Gln). The arrangement of these genes on the physical map of T5 phage was as follows: -tRNA^Gln-tRNA^His-RNA III-RNA I-…-tRNA^Asp.     

Characterization of histones and chromatin of the hepatopancreas in Palaemon serratus (Crustacea Natantia)]

The chromatin of shrimp hepatopancreas has been extracted from isolated nuclei and characterized. Nuclei were prepared in the presence of Cu++ and phenyl methyl sulfonyl fluoride in order to inhibit the nuclease and protease activities throughout the different purification steps. The purified nuclei are heterogenous in size and show a density of 1,367 g/ml determined on saccharose - glucose gradients. After washing in 0,14 M NaCl and then in 10(-2) M Tris-HCL, pH = 7,6, the nuclei were disrupted in water. The solubilized chromatin was precipitated in 0,15 M.NaCl. This chromatin is characterized by a high level of RNA (RNA/DNA = 0,38) and of non histone proteins (NHP/DNA = 0,6). The denaturation curve showed only one Tm at 69 degrees in 2.10(-4) M.EDTA. When the chromatin was extracted in the presence of staphylococcal nuclease, the Tm reached 80 degrees C. The kinetics of the digestion by the staphylococcal nuclease have been studied and show that 10 per cent of hydrolysis occurs within the first minute. The repeat length of DNA as determined with the polymers of higher order is 189 +/- 5 base pairs. The existence of nucleosomes was confirmed by electron microscopy. The superstructure of chromatin was not completely destroyed after solubilisation with a Potter. The histones were studied by gel electrophoresis after differential staining. The most important feature consists in the presence of two H1, two H2A and two H4. The acetylation levels of the histones were followed after injection of 14C-acetate in vivo. The subfraction H1, 0 was acetylated. Only one H3 was present and the two H2A fractions showed the same level of acetylation. H2B migrated faster than the H2A fractions like in Echinoderms. The two H4 fractions corresponded to two differently acetylated forms. Shrimp hepatopancreas histones were fractionated by molecular sieving on Biogel P 100 and characterized according to their electrophoretic properties as well as their amino-acid content. The amino-acid compositions of the different histone fractions were nearer to Echinoderm and Sipunculid histones, than Calf thymus homologue histones. All the fractions show a weaker basicity. The H3 fraction was the only one showing a lesser variability when compared to Calf thymus H3. The non histone proteins were extracted in 10(-2) M Tris-HCL, pH = 8 and 0.1 per cent SDS. A series of 50 proteins was detected. 80 per cent of the total amount of protein was localized in a molecular weight range comprised between 40 000 and 80 000 daltons. These proteins were compared to the histones and total proteins of sonicated chromatin solubilized by SDS in order to detect proteasic effects.