Centrifugation of homogenates of bovine splanchnic nerve trunks and adrenal medulla showed that 47% and 31%, respectively, of the acetylcholinesterase activity was not sedimentable. The possibility that the soluble acetylcholinesterase activity was derived artefactually from the membrane-bound form was excluded. Gel electrophoresis revealed that the soluble acetylcholinesterase activity in adrenal medulla was distributed between five isoenzymes, only one of which was found in the supernatant fraction of splanchnic nerve homogenates. Solubilization of the membranes of both tissues with Triton X-100 gave a single isoenzyme of acetylcholinesterase, common to both tissues, whose electrophoretic mobility was less than that of any of the soluble isoenzymes. Some of the properties of the soluble and membrane-bound isoenzymes of acetylcholinesterase of splanchnic nerve were compared: the Michaelis constants were identical, both isoenzymes sedimented to the same position as catalase (molecular mass 240000) in a density gradient, their mobilities varied in a parallel fashion upon electrophoresis in gels of different concentration. It was concluded that the higher electrophoretic mobility of the soluble isoenzyme was because it carried a greater charge than, but had a similar molecular mass to, the membrane-bound isoenzyme.

Isoenzymes of soluble and membrane-bound acetylcholinesterase in bovine splanchnic nerve and adrenal medulla

Different forms of acetylcholinesterase (AChE), EC 3.1.1.7, were demonstrated in human brain caudate nucleus. One form was solubilized at high ionic strength, the other with Triton X-100. The detergent-extractable form was purified to homogeneity by affinity chromatography. This form of AChE is amphiphile-dependent; i.e., it was active only in the presence of amphiphiles (detergents or lipids). Further, the enzyme was shown to bind detergents and to interact hydrophobically with Phenyl-Sepharose. In the presence of detergents the enzyme is a tetramer (subunit molecular weight, 78,000) which aggregates on the removal of detergents. Human brain AChE showed a reaction of identity with human erythrocyte AChE in crossed-line immunoelectrophoresis. The high-salt-soluble brain enzyme did not cross-react with the erythrocyte enzyme. The two classes of AChE seem not to be related, as they show no common antigenic determinant.

An Amphiphile‐Dependent Form of Human Brain Caudate Nucleus Acetylcholinesterase: Purification and Properties

Summary

1. In this review, representative data on the nature of enzyme multiplicity and the developmental progressions of multiple enzyme forms have been collected, and the significance of this material has been discussed in relation to gene involvement during tissue differentiation.



2. In addition, emphasis has been directed towards the relevance of this onto-genetic information to a wide variety of biological phenomena, such as the possibilities of metabolic advantage conferred by the distinctive properties and physiological locations of these multiple enzyme forms, the insight into the subunit structure and compositional interrelationships of proteins, the aetiology of disease states, the relationship to hormone action, the extra nuclear specification of enzymes, and the transformations between particulate and soluble enzymes.



3. Three isoenzyme systems in particular have been chosen for the special development of these themes and treated in some detail; lactate dehydrogenase (LDH), aldolase and the esterases. Briefer accounts of the ontogeny of other less-studied series of multiple enzyme forms have also been included.



4. In relation to LDH, the major findings and conclusions of the pioneering investigators have been outlined, together with the reassessments which have been necessitated by recent studies of LDH ontogeny. It is evident, for example, that the early embryonic form of this enzyme is not necessarily parental as previously thought, and the realization appears to possess wide-ranging implications in regard to the control of synthesis of the enzymes. An indirect role of oxygen in LDH biosynthesis is indicated together with the independent regulation of the separate genes.



5. Recent developments in the enzymology of preimplantation ova have been discussed. It is noteworthy that the extraordinary levels of LDH activity which have been reported to be associated with these early developmental stages, may arise by extracellular adsorption rather than by an intrinsic synthesis of enzyme. Hence contemporary interest in this area has shifted towards a comprehension of the biochemical significance of the extremely unusual, selective concentrations of extracellular lactate dehydrogenase in the oviduct, the relation of this enzyme to the specialized growth requirements of the ova, and the influences of reproductive hormones on these oviducal isoenzymes.



6. The role of aldolase ontogeny as well as the composition, gene control, and native state of these lysases is also reviewed. This enzyme provides a particular example of the manner in which tissue modification of the catalytic and structural properties of enzymes may influence interpretations of the native state of purified proteins, and in this case, also, the observable microheterogeneity satisfies previous disquietude in regard to the existence of different subunits in the muscle enzyme.



7. The basic developmental importance of aldolase-C is stressed by the occurrence of this form of the enzyme in the early embryos of many different species. Furthermore, a significant addition to our knowledge of the possible biological purposes of isoenzymes and the rationale of their ontogenetic development during the establishment of tissue identity would appear to follow from the recent studies on reversible adsorption of aldolase to cell membranes. Aldolase-C. for example, is differentially inhibited in this way by the end product of anaerobic glycolysis, and other such metabolite-isoenzyme-tissue interactions allow for a wide range of tissue-specific responses regulating energy derivation and other important functions of the cell.



8. The remarkable extent of heterogeneity among the esterases, the distinctive species-specific nature of their multiplicity, and the overlapping substrate specificities have long presented a formidable barrier to the unravelling of the isoenzyme status of these enzymes. The advantages of systematic approaches utilizing differential substrate and inhibitor specificities have been emphasized, and the most recently available information summarized.



9. The carboxylesterases may be divided into five distinct groups, and recognizable subdivisions also occur within the other classes of esterases. Most of the different possibilities of isoenzyme causation are exemplified within the esterases, and the ontogenetic specificity of their heteromorphs seems to be greater than for any other recorded isoenzymic system.



10. In addition to the generative features displayed by the ‘soluble’ enzyme forms, the potential utility of latent isoenzyme activities have been illustrated by reference to catalase. The properties of such systems provide a useful means of studying such problems as the subcellular specification of enzyme synthesis and the nature of the mechanism governing the distribution of specific proteins between organelles and cytosol.



11. Catalase also provides a situation of special interest in that the existence of isoenzymes and even of activity in the cytosol have been disputed until very recently. Evidence establishing the reality of these occurrences, and the epigenetic nature of catalase multiplicity in some species, has been cited.



12. Recently available ontogenetic information of a number of oxidoreductases, transferases and hydrolases has also been collected together and discussed.

Isoenzymes and ontogeny.

The Postnatal Development of the Human Cerebral Cortex

The ontogeny of the esterase isozymes of the teleost, Fundulus heteroclitus, has been investigated. One group of esterase isozymes is present at all stages of development, whereas other esterase isozymes only very gradually appear at later stages of development, or abruptly appear at such dramatic developmental events as hatching. The ontogeny of these isozyme patterns is interpreted as the expression of differential regulation of separate esterase genes. The general pattern of teleost esterase gene activation is similar to that reported for birds and mammals. Allelic variation was detected at two of the esterase loci. On the basis of electrophoretic mobility, substrate specificity, inhibitor specificity, genetic variation, and ontogeny of esterases, there appear to be at least 15 different esterase isozymes, which constitute 6–8 groups, each of which is probably encoded in one or more genetic loci.

Developmental genetics of the esterase isozymes of Fundulus heteroclitus

The free and bound nonspecific esterases occurring in, respectively, the saline and triton X-100 extracts of adult and developing human brain were studied by starch gel electrophoresis (zymograms). Zymograms of the free esterase fraction visualized with NA as substrate were qualitatively similar at 5-12 days of age to the electrophoretic patterns observed in adult material. In both adult and developing brain, zymograms of bound esterase resembled those of the free enzyme, the major difference being the presence in the former of a slow, broad, anodic zone of diethyl-p-nitrophenyl phosphate-inhibited enzyme.



Esterases characteristic of adult white matter and having preferential affinity for alpha-naphthylpropionate, alpha-naphthyl butyrate and alpha-naphthyl valerate were not identified in infant brain until about 4 months postnatal age.



A far-moving, anodic enzyme was distinctly evident in zymograms of brains of less than 1 month of age. This enzyme hydrolysed NP, NB, and NV more actively than alpha-naphthyl acetate. It was present in the adult brain but, in contrast to the infant, was no longer electrophoretically-separable from another enzyme which had greater affinity for NA and had previously been designated the A10 band.



Quantitative assays demonstrated that the bound esterase increased in cerebral and cerebellar cortex during development. In contrast, the proportion of free to bound esterase showed little change in white matter.



Acetylcholinesterase and butyrylcholinesterase zymograms became identical to adult patterns from 1 to 4 months of age.



Thiolacetic acid esterase was present at 38 weeks gestation.



Some functional and anatomical correlations were attempted in explanation of the biochemical observations.

K. D. Barron

Papers

Esterases of developing human brain