Extraction and immunochemical assays of a tubulin-like factor in cotton seedlings

Abstract

Antibodies to tubulin were prepared in rabbits by immunization with reduced-carboxymethylated calf-brain tubulin. In immunodiffusion tests the antibodies showed full cross reactivity with the immunogen as well as with native calf-brain tubulin. The same antibodies showed cross reactivity with a factor in extract of cotton (Gossypium hirsutum L.) cotyledons but there was no full immunological identity between calf-brain tubulin and this factor. A solid-phase radioimmunoassay for quantitative estimation of this plant tubulin-like factor was developed. It measured the binding of antibodies to immobilized calf-native tubulin. Competition between the unknown soluble tubulin-like factor, and immobilized tubulin was assayed at serum dilution of 1:50. Extraction conditions which preserved the antigenic properties of the tubulin-like factor from cotton cotyledons were defined. The radioimmunoassay measured quantities of the tubulin-like factor in the range of 0.1-10 μg-equivalents of calf-brain tubulin. Immediately after homogenization of the tissue only 25% of the total amount of tubulin-like activity was present in soluble form, while most of it remained in the insoluble fraction. Apparent maximal solubilization was achieved spontaneously 10 h after homogenization or by treatment with guanidine hydrochloride. These results indicate that in this material, tubulin is not released immediately by homogenization but remains assembled in microtubules and-or in a bound or sequestered form.

Quantitation of Chill-Induced Release of a Tubulin-Like Factor and Its Prevention by Abscisic Acid in Gossypium hirsutum L.

“…After centrifugation, the supematant was dialyzed against the extraction medium without guanidine-HCl. This procedure is assumed to result in complete release of all the tubulin-like factor (16). The amount of free tubulin-like factor is expressed as percentage of the total amount of tubulin-like factor.…”

“…The homogenate was immediately centrifuged at 100,000g for 45 min. The amount of the tubulin-like factor in the supernatant was measured by radioimmunoassay and expressed as pg-eq of calf brain tubulin as earlier described by Rikin et al (16). The total amount of tubulin-like factor in the tissue was determined by the radioimmunoassay after extraction under the previous conditions, but the extraction medium contained 0.5 M guanidine-HCl, and the homogenate was kept at room temperature for 30 min.…”

“…In the present study, we attempted to obtain more direct evidence for the role of microtubules in development of chilling injury by measuring the degree of tubulin polymerization. The degree of tubulin polymerization in the tissue was estimated by a recently developed radioimmunoassay which measured the amount of a tubulin-like factor (16).…”

Tubulins from different higher plant species are immunologically nonidentical and bind colchicine differentially

“…When each IgG was used at a dilution of 1:100, the autoradiographic band intensity produced by the a-subunit IgG (a- The cross-reactivity of antibodies raised against animal tubulin with tubulin-like factors in plant extracts (5,6) and with microtubules in plant cells (7)(8)(9) variability among the a-subunits than among the /3-subunits ( Fig. 2A) (Fig.…”

“…The main structural component of microtubules is tubulin, a conserved heterodimeric protein having a molecular weight of 100,000 (2)(3)(4) and composed of one asubunit and one 3-subunit (Mr, -50,000 each). Antibodies raised against animal tubulin have been shown to bind to tubulin-like components of plant cell extracts (5,6) and to decorate microtubules by indirect immunofluorescence (7,8) or immunogold staining (9). This cross-reactivity of animal tubulin antibodies has suggested that plant and animal tubulins are immunologically similar.…”