The effect of some injurious agents on the biochemistry of brain development and a study of some characteristics of synaptic membrane acetylcholinesterase

Abstract

The effects of maternal hyperthermia on the state of development of newborn guinea-pig brain have been studied with particular regard to DNA content and the activities of brain plasma membrane marker enzymes. Hyperthermia of about 4 C applied to pregnant guinea-pigs daily between the l8th and 25th days of gestation resulted in newborn exhibiting a high degree of microencephaly, but with no other obvious abnormalities. Microencephalic animals were also found to have reduced brain DNA content. However, the reduction in brain weight and DNA content were localised primarily to the cerebral hemispheres, as no such effects were detected for cerebellar tissue. The protein specific activities of plasma membrane marker enzymes from synaptic and microsomal membrane fractions from the brains of microencephalic newborn were found to be similar to those from control animals. On the other hand, the activities of these enzymes in homogenates of microencephalic guinea-pig cerebral tissue, when expressed as a function of DNA content, have been shown to be higher than such values for control cerebral tissue. These results have been discussed in terms of current models of brain development, and it is suggested that heat damage in the earliest stages of brain development can influence the subsequent differentiation of brain cells. In this case it is suggested that hyperthermia indirectly brings about more elaborate dendritic development in surviving brain cells. The effects of hypothyroidism on the development of the specific activities of synaptic membrane enzymes in the rat brain has also been investigated. In control rats the specific activities of synaptic plasma membrane ATPases and acetylcholinesterase increased in the immediate postnatal period. The rates of increase in the activities of these enzymes in membrane preparations from hypothyroid rat brain were lower than for control preparations after about the 10th or 12th days of gestation, and did not attain normal adult values by 44 days post parturn. These lower enzyme activity values have been shown not to be due to an increase in the 'latent' enzyme fraction hidden within membrane vesicles. This description of qualitative changes in the development of synapses in rat brain has been discussed in terms of current models of brain dysfunction in hypothyroid rat brain which ascribe significance only to quantitative reduction in brain cell growth. Some characteristics of synaptic membrane acetylcholinesterase from normal rat brain have also been examined in order to evaluate the possible role for lipid in the properties of this enzyme. Non-linear Arrhenius temperature-activity plots, usually associated with lipid dependent enzymes, have been found for acetylcholinesterase. The effects of membrane association, detergents and other lipophilic agents on the activity and kinetic properties of acetylcholinesterase have been evaluated. These data have been interpreted as evidence for a lipoprotein structure for acetylcholinesterase and for the involvement of lipid in the properties of this enzyme, particularly in the maintenance of a low activation energy state at physiological temperatures. This lipid has been tentatively identified as cardiolipin (diphosphatidylglycerol) as a phospholipid extracted from partially purified acetylcholinesterase in high salt conditions migrated on two dimensional thin-layer chromatography plates in a similar fashion to a commercial cardiolipin standard. The low-ionic strength soluble and membrane-bound fractions of synaptic membrane acetylcholinesterase were further examined in order to assess the role of the membrane in enzyme properties. The membrane-enzyme was shown to be much more stable to irreversible thermal inactivation than the soluble. However, this difference was also observed in Lubrol detergent solubilized preparations. The difference in stability of these fractions has in this case been attributed not to membrane association, but to differences in the oligomeric nature of soluble and membrane- bound acetylcholinesterase. These properties of acetylcholinesterase have been discussed in terms of a potential model for lipid-protein interactions in general.