Biomedical subjects
H Byrne
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Psychiatric disturbance in children.
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Subcellular localization of cellulases in auxin-treated pea.
Two forms of cellulase, buffer soluble (BS) and buffer insoluble (BI), are induced as a result of auxin treatment of dark-grown pea epicotyls. These two cellulases have been purified to homogeneity. Antibodies raised against the purified cellulases were conjugated with ferritin and were used to localize the two cellulases. Tissue sections were fixed in cold paraformaldehyde-glutaraldehyde and incubated for 1 h in the ferritin conjugates. The sections were washed with continuous shaking for 18 h and subsequently postfixed in osmium tetroxide. Tissue incubated in unconjugated ferritin was used as a control. A major part of BI cellulase is localized at the inner surface of the cell wall in close association with microfibrils. BS cellulase is localized mainly within the distended endoplasmic reticulum. Gogli complex and plasma membrane appear to be completely devoid of any cellulase activity. These observations are consistent with cytochemical localization and biochemical data on the distribution of these two cellulases among various cell and membrane fractions.
Purification and characterization of two cellulases from auxin-treated pea epicotyls.
Two forms of beta-1,4-glucan 4-glucanohydrolase (EC 3.2.1.4) were extracted from growing regions of Pisum sativum epicotyls which had been treated with the auxin, (2,4-dichlorophenoxy)acetic acid. One cellulase is buffer-soluble, the other buffer-insoluble but extractable with high salt concentrations. Both enzymes catalyze endohydrolysis of carboxymethylcellulose with the same pH optimum (5.5 to 6.0). They were purified with the use of DEAE-cellulose chromatography, Sephadex gel filtration, and ultrafiltration. They are distinct proteins as characterized by: electrofocusing and disc gel electrophoresis (pI values = 5.2 and 6.9, respectively); mobility in sodium dodecyl sulfate polyacrylamide gels, fractionation on Sephadex, and sedimentation in the ultracentrifuge (mol wt = approximately 20,000 and 70,000, S values 2.63 and 3.73); and immunological properties. The buffer-soluble enzyme tends to dimerize on purification. Amino acid analyses show that the buffer-soluble enzyme is relatively rich in glycine, alanine, and valine and deficient in cystine, tryosine, and phenylalanine compared to the buffer-insoluble enzyme. The two cellulase activities were generated in approximately equal amounts after auxin treatment. Within 5 days their levels had increased at least 100-fold and they constituted about 0.1% of total cellular protein. Present data indicate that one is not derived from the other.
Regulation and in vitro translation of messenger ribonucleic acid for cellulase from auxin-treated pea epicotyls.
Polysomal RNA was isolated from pea epicotyls treated with (2,4-dichlorophenoxy)acetic acid, and fractionated on oligo(dT)-CELLULOSE TO YIELD POLY(A)-containing RNA. This RNA fraction was translated in a wheat embryo cell-free system and found to have more than 90% of the messenger activity in total polysomal RNA. Immunoprecipitation of the translation product by monospecific antibodies to pea cellulases (beta-1,4-glucan 4-glucanohydrolase, EC 3.2.1.4) indicated that cellulase was synthesized in this system. The immunoprecipitate co-migrated with the buffer-soluble cellulase component in sodium dodecyl sulfate-gel electrophoresis. Buffer-insoluble cellulase was not detected in the in vitro translation products. Fractionation of mRNA from membrane-bound and free polysomes and their subsequent translation indicated preferential synthesis of buffer-soluble cellulase on membrane-bound polysomes. With the above techniques for assaying buffer-soluble cellulase mRNA, a 10-fold increase in the level of this messenger per tissue segment was observed within 48 hours following (2,4-dichlorophenoxy)acetic acid treatment. There was no evidence for pre-existing untranslated message for cellulase in control tissues. Since there was no delay in the appearance of mRNA for cellulase, compared to a 24-hour lag in the increase of cellulase activity, it is suggested that translational as well as transcriptional controls are exerted on the biosynthesis of cellulase in vivo. Analysis of the rates of peptide chain initiation and elongation, both in vivo and in vitro, indicated that peptide chain elongation may be rate-limiting during the lag phase of cellulase development.