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Effect of Hypothyroidism on Different Forms of Actin in Rat Cerebral Neuronal Cultures Studied by an Improved DNase I Inhibition Assay
Author(s) -
Paul Surojit,
Das Sumantra,
Sarkar Pranab Kumar
Publication year - 1992
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1992.tb09425.x
Subject(s) - actin , cytoskeleton , lysis , actina , actin cytoskeleton , biology , microfilament , biochemistry , chemistry , microbiology and biotechnology , cell
An improved DNase I inhibition assay for the filamentous actin (F‐actin) and monomeric actin (G‐actin) in brain cells has been developed. Unlike other methods, the cell lysis conditions and postlysis treatments, established by us, inhibited the temporal inactivation of actin in the cell lysate and maintained a stable F‐actin/G‐actin ratio for at least 4‐5 h after lysis. The new procedure allowed separate quantitation of the noncytoskeletal F‐actin in the Triton‐soluble fraction (12,000 g, 10 min supernatant) that did not readily sediment with the Triton‐insoluble cytoskeletal F‐actin (12,000 g, 10 min pellet). We have applied this modified assay system to study the effect of hypothyroidism on different forms of actin using primary cultures of neurons derived from cerebra of neonatal normal and hypothyroid rats. Our results showed a 20% increase in the Triton‐insoluble cytoskeletal F‐actin in cultures from hypothyroid brain relative to normal controls. In the Triton‐soluble fraction, containing the G‐actin and the noncytoskeletal F‐actin, cultures from hypothyroid brain showed a 15% increase in G‐actin, whereas the F‐actin remained unaltered. The 10% increase in total actin observed in this fraction from hypothyroid brain could be totally accounted for by the enhancement of G‐actin. The mean F‐actin/G‐actin ratio in this fraction was about 30% higher in the cultures from normal brain compared to that of the hypothyroid system, which indicates that hypothyroidism tends to decrease the proportion of noncytoskeletal F‐actin relative to G‐actin.