A radiolabeled monoclonal antibody binding assay for cytoskeletal tubulin in cultured cells

To detect changes in the extent of tubulin polymerization in cultured cells, we have developed a radioactive antibody binding assay that can be used to quantitate total cytoskeletal tubulin or specific antigenic subsets of polymerized tubulin. Fibroblastic cells, grown to confluence in multiwell plates, were permeabilized and extracted with 0.5% Triton X-100 in a microtubule-stabilizing buffer. These extracted cytoskeletons were then fixed and incubated with translationally radiolabeled monoclonal antitubulin antibody (Ab 1-1.1), an IgM antibody specific for the beta subunit of tubulin. Specific binding of Ab 1-1.1 to the cytoskeletons was saturable and of a single apparent affinity. All specific binding was blocked by preincubation of the radiolabeled antibody with excess purified brain tubulin. Specific Ab 1-1.1 binding appeared to represent binding to cytoskeletal tubulin inasmuch as: pretreatment of cells with colchicine decreased Ab 1-1.1 binding in a dose-dependent manner which correlated with the amount of polymerized tubulin visualized in parallel cultures by indirect immunofluorescence, taxol pretreatment alone caused an increase in Ab 1-1.1 binding and prevented in a dose-dependent manner the colchicine-induced decrease in antibody binding, in cells pretreated with colcemid and returned to fresh medium, Ab 1-1.1 binding decreased and recovered in parallel with the depolymerization and regrowth of microtubules in these cells, and comparison of maximal antibody binding per cell between primary mouse embryo, 3T3, and human foreskin fibroblasts correlated with immunofluorescence visualization of microtubules in these cells. Thus, this assay can be used to measure relative changes in the level of polymerized cytoskeletal tubulin. Moreover, by Scatchard-type analysis of the binding data it is possible to estimate the total number of antibody binding sites per cell. Therefore, depending on the stoichiometry of antibody binding, this type of assay may be used for quantitating total cytoskeletal tubulin, specific antigenic subsets of cytoskeletal tubulin, or other cytoskeletal proteins.     

General recombination mechanisms in extracts of meiotic cells

RecA-like proteins have been purified from somatic and meiotic cells of mouse and lily. The rec proteins have been designated s-rec and m-rec to indicate their respective tissues of origin. The two proteins differ in molecular weight and in their response to temperature, the latter being consistent with the optimal temperature for physiological function of their tissues of origin. There is a major increase in m-rec protein with the entry of cells into meiosis, the peak of activity being early pachytene. Extracts of the cells and also those of yeast (Saccharomyces cerevisiae) have been prepared that have the capacity to catalyze homologous recombination. These extracts behave similarly to the m-rec proteins upon entry of cells into meiosis. Yeast transferred to sporulation medium displays a 100-fold increase in the recombination activity of the extract at about the time of entry into meiosis. The occurrence of peak levels of m-rec and recombination activity in extracts from cells in early pachytene points strongly to that stage as the time at which the enzymatic phase of recombination occurs.     

Level of translatable messenger RNA coding for argininosuccinate synthetase in the liver of the patients with quantitative-type citrullinemia

Summary The translation activity of mRNA coding for argininosuccinate synthetase in total RNA extracted from the liver of three patients with quantitative-type citrullinemia was determined using a cell-free translation system. In two patients, the hepatic content of the enzyme was about 20% of the control value, whereas translatable mRNA level for the enzyme was similar to or slightly lower than those of control livers. In the third patient, the enzyme content was about 50% of the control value, and mRNA activity for the enzyme was low normal. These results indicate that at least in the first two patients, the decrease in the enzyme protein is due either to increased degradation of the enzyme or to decreased translation in the patient's liver.     

Stereospecific incorporation of hydrogens from NADPH in elongation of very long fatty acyl-CoA by swine cerebral microsomes

The elongation of arachidoyl-CoA by swine cerebral microsomes resulted in the production of behenic acid (22:0) and lignoceric acid (24:0) concomitantly. When 4S-[4-²H₁]NADPH was used for the elongation of arachidoyl-CoA, the incorporation of two deuterium atoms into 22:0 was observed by the technique of mass fragmentography. Furthermore, the incorporation of four deuterium atoms into 24:0 was also detected. On the other hand, when 4R-[4-²H₁]NADPH was used, no deuterium was incorporated into the elongated products.     

Heterogeneity among microtubules of the cytoplasmic microtubule complex detected by a monoclonal antibody to alpha tubulin

Three monoclonal antibodies specific for tubulin were tested by indirect immunofluorescence for their ability to stain cytoplasmic microtubules of mouse and human fibroblastic cells. We used double label immunofluorescence to compare the staining patterns of these antibodies with the total microtubule complex in the same cells that were stained with a polyclonal rabbit antitubulin reagent. Two of the monoclonal antitubulin antibodies bound to all of the cytoplasmic microtubules but Ab 1-6. 1 bound only a subset of cytoplasmic microtubules within individual fixed cells. Differential staining patterns were observed under various fixation conditions and staining protocols, in detergent-extracted cytoskeletons as well as in whole fixed cells. At least one physiologically defined subset of cytoplasmic microtubules, those remaining in cells pretreated for 1 h with 5 microM colcemid, appeared to consist entirely of Ab 1-6. 1 positive microtubules. The same was not true of the microtubules that remained in either cold-treated cells or in cells that had been exposed to hypotonic medium. The demonstration of antigenic differences among microtubules within single fixed cells and the apparent correlation of this antigenic difference with at least one "physiologically" defined subset suggests that mechanisms exist for the differential assembly or postassembly modification of individual microtubules in vivo, which may endow them with different physical or functional properties.     

CO2 is the first species formed upon CO oxidation by CO dehydrogenase from Pseudomonas carboxydovorans

The active species of CO₂, i.e. CO₂ or HCO ₃ ^- , formed in the CO dehydrogenase reaction was determined using the pure enzyme from the carboxydotrophic bacterium Pseudomonas carboxydovorans. Employing an assay system similar to that used to test for carbonic anhydrase, data were obtained which are quite compatible with those expected if CO₂ is the first species formed. In addition, carbonic anhydrase activity was not detected in P. carboxydovorans.     

Do protozoa conceal a high potency of nucleoside triphosphate hydrolysis present in Toxoplasma gondii?

ATP hydrolytic activity in whole cell homogenates of some protozoa was assayed in the presence or absence of dithiothreitol. The activities in all protozoan cell homogenates, except Toxoplasma gondii, ranged from 0.6 to 32 mumol/mg protein/hr, irrespective of the presence or absence of dithiothreitol. A remarkably higher activity, 11,690 mumol/mg protein/hr, was observed for T. gondii in the presence of dithiothreitol. These results indicate that the higher ATP hydrolytic potency observed for T. gondii is not universal to protozoa, rather it is unique to T. gondii.     

Photoreception in pineal organs of larval and adult lampreys,Lampetra japonica

A comparative study of the larval and adult pineal organs, which are sensitive to incident light, was carried out in the river lamprey Lampetra japonica, using intracellular recording from the pineal photoreceptors. The tissue overlying the larval pineal organ is transparent, whereas that over the adult pineal is translucent. The optical density of this oval pineal window in the adult lamprey was 1.2. In order to elucidate the early development of the larval pineal, the ratio r of the diameter (micron) of the pineal to the body-length (cm) was measured. The value of r was 62.5 in a small larva of 2.8 cm, 29.7 in a larger one of 14.3 cm, and 9.3 in an adult of 54 cm body-length. The intracellular response to light of the larval pineal was a hyperpolarization, showing fundamentally the same pattern as that of the adult pineal. It was possible to record a typical response even from the pineal of the smallest larva, 2.8 cm in body length, used in this study. The intensity-amplitude relationship was analysed after Naka-Rushton's hyperbolic equation. The value of sigma of isolated larval pineals was 0.88 log unit higher than that of adults. The value of n was larger in larvae, suggesting a sensitive reaction to changing photic stimulus. The spectral sensitivity was compared. The peak was at 505 nm in the larva, but 525 nm in the adult. A change of visual pigment in the pineal during metamorphosis is suggested.     

Electrophoretic heterogeneity and age-related change of serine proteinase fromAspergillus sojae

Five forms of serine proteinase (EC 3.4.21.14) have been purified fromAspergillus sojae. This paper reports heterogeneity of electrophoretic mobilities, isoelectric points, and kinetic parameters of multiple forms of serine proteinase fromAspergillus sojae, including agerelated changes in thek _cat /K _m of electrophoretic species.