Adeline R. Hess

SUBCELLULAR LOCALIZATION OF RAT BRAIN ESTERASES

The localization and properties of soluble and bound esterases of subcellular fractions of rat brain have been investigated. Bound esterases were extracted with 1% Triton X-100 and separated by starch gel electrophoresis. By these means a molecular population of isoenzymes was demonstrable that was quantitatively different from the isoenzyme population of the watersoluble esterase activity. The highest specific activity for α-naphthyl acetate hydrolysis was contained in the microsomal fraction and could be extracted by Triton. In contradistinction to whole brain and to other subcellular fractions, microsomes contained a molecular population of esterase isozymes which was qualitatively distinct from that of water extracts in that the fast moving A-type esterases were absent. In addition, there was present a heavy concentration of slow movinig B-esterase. Acetylcholinesterase could also be extracted from this fraction by Triton, migrated with B-esterase and actively hydrolyzed αnaphthyl acetate. Combined electron microscopic and quantitative chemical analysis of the subcellular fractions suggested that some bound nonspecific esterase may be localized in sub-synaptic membranes. The pI50 values for E 600 of the soluble and insoluble, Triton X-100-extracted and Triton X-100-insoluble esterases are, respectively, 5.3, 7.5, 7.5 and 7.4. It is noted that these results may be determined in part by the participation of acetylcholinesterase in hydrolysis of the substrate. Mitochondria are virtually devoid of esteratic activity. C-type esterase (CMB-activated, E 600-resistant) occurred in both the bound and soluble state. A-, B- and C-type esterases exist in both bound and soluble forms and their chemical properties appear to be independent of their site of localization. Histochemical studies indicate that the B-type esterase is localized predominantly to cytoplasm of neurons and to neuropil. A-esterase is localized to droplets (presumed lysosomes) of neurons and other cell types, e.g., pericytes. The histochemical localization of C-esterase could not be determined.

SEPARATION AND PROPERTIES OF HUMAN BRAIN ESTERASES

hlhoresis. 2. From 20 to 25 water-soluble proteins can ho’ visualized on the gels by application of a simple amidoblack 1011 staining procedure. Within ami individual brain, these Proteins present the same basic pattern whatever the region samhlled. 3. By the zvmogram technique at least 18 bands active against naphthol esters can be separated. Hydrolysis of esters of o’hain length gr(’at(’r than the butyrate is not demonstrable. 4. In homogenates of caudate-putamen, dcctr(IIIh(Iresis and use of thiocholine ester substrate solutions demonstrate 3 bands having properties of acetvlchohnesterases. These do not hydrolyze non-choline esters. 5. Indoxvl and naphthvl acetates appear to be tiy(lrolvzed by the same enzymes. 6. The major activity against thiolacetic acid is concentrated in a 1)alid on the far cathodal side, at a position relatively remote from the other enzyme activities mentioned.

Proteins and isozymes of esterases and cholinesterases from sera of different species

Abstract Serum proteins of the human, cat, rabbit, monkey and rat were separated by starch gel electrophoresis and in addition to protein, cholinesterase, esterase and proteolytic activities were determined. In human serum, as many as 7 esterases were visualized with thiocholine esters. The major band of activity was only partially sensitive to physostigmine. No abnormal cholinesterase pattern was apparent in 7 cases of myasthenia gravis. The patterns of esterase activities in the species studied showed conspicuous differences. Except in the monkey, some activity was associated with the albumin fraction. In human sera, with α-naphthyl propionate and a-naphthyl butyrate as substrates, a pre-albumin band of activity could be elicited. This was not present when α-naphthyl acetate was used. In different species, the resistance and sensitivity of similar enzymes to the inhibitors employed was highly variable. In human serum proteolytic activity developed with N-benzoyl-dl-arginine-β-naphthylamide included at least 2 zones of activity which did not react with thiocholine and naphthyl esters. The partial inhibition by physostigmine of the major band of activity developed in human serum with thiocholine and naphthyl esters is noted and its significance is discussed.

ALTERATIONS IN PROPERTIES AND ISOENZYME PATTERNS OF ESTERASES IN DEVELOPING RAT BRAIN

NUMEROUS studies of chemical and enzymic changes in the brain during maturation have been reported. One objective of these studies was t o correlate such chemical and enzymic changes with morphological and functional alterations in the developing central nervous system. The inherent complexity of the problem occasioned by the occurrence of different cell types and structures in the brain has been increased by the finding that enzymes may exist in multiple molecular varieties. Electrophoresis of human brain material on starch gel results in the separation of as many as 2G-25 bands exhibiting esterase activity (BARRON et al., 1963). There appear to be three acetylcholinesterases in human caudate nucleus and putamen (BERNSOHN et al., 1962). At the present time, the nature of the differences between the molecular species responsible for the varying rates of migration in starch gels under the influence of an electric field is not clear. SVENSMARK (1961) has shown that butyrylcholinesterase has sialic acid as a conjugate which, when liberated by neuraminidase, alters the mobility of the enzyme, and WIELAND and PFLEIDERER (1961) have reported varying ratios of different amino acids in lactic acid dehydrogenase isoenzymes. 7 The development of the zymogram technique by HUNTER and MARKERT (1957) and our interest in brain esterases has prompted an investigation into the differentiation of esterase isoenzymes in the developing rat brain. In addition to the information obtained on the esterase activity of the developing brain, the present study has provided further insight into the nature of an isoenzyme series.

ZYMOGRAMS OF NEURAL ACID PHOSPHATASES. IMPLICATIONS FOR SLIDE HISTOCHEMISTRY.

Fi;. 5. (left) Front the parenchymal cell of a rabbit liver after 60 minutes incubation in the ATP me(hum. Activity is prominent in patches of apparently rough surfaced ER. There is probably spurious st ailting of nuclear cliroinat in. X 17,400. FI(;. 6. ( right ( 11 igher magnificat ion from an area shown in Fig. 5. Cisternae are empty hut there is striking act ivitv in their limiting menibranes. X48000.

STARCH GEL ELECTROPHORESIS OF BRAIN ESTERASES

1. Starch gel electrophoresis of rat brain homogenates has demonstrated 9 esterases which hydrolyze α-naphthyl acetate and naphthol AS-acetate. One of these has been identified as acetylcholinesterase. The nature of the others is undefined. In addition an enzyme (presumably pseudocholinesterase) acting against butyrylthiocholine iodide has been located at the site of sample insertion. 2. Homogenates of human white matter have yielded 9 esterases active against the same naphthol substrates. None of these is a cholinesterase. However, with acetylthiocholine iodide as substrate, acetylcholinesterase is demonstrable in human material. Enzymic activity against butyrylthiocholine iodide is demonstrable at the origin in human material, also. 3. Best results are obtained by use of homogenates centrifuged at 20,000 x g for 60 minutes.

Cholinesterases in serum as demonstrated by starch gel electrophoresis.

Summary As many as 7 bands of enzymatic activity against choline esters are demonstrable in human, rat and cat sera by starch gel electrophoresis. The evidence suggests that these bands derive from distinct molecular species of enzyme. However, possible influences on “zymogram” patterns of enzyme adsorption by inert protein require investigation.

CHOLINESTERASES AND NONSPECIFIC ESTERASES OF DEVELOPING AND ADULT (NORMAL AND ATROPHIC) RAT GASTROCNEMIUS 1. CHEMICAL ASSAY AND ELECTROPHORESIS

Cholinesterases and nonspecific esterases of normal and atrophic (denervated and tenotomized) adult rat gastrocnemius were assayed, the latter 5-45 days after operation. Acetylcholinesterase activity per whole muscle was about 25% of normal 5 days after denervation and, in permanently denervated animals, showed little change thereafter. Tenotomy caused a transient 27% fall. Butyrylcholinesterase was unaffected. Nonspecific esterase activity of denervated gastrocnemius (substrate, α-naphthyl acetate) was normal 30 days after reversible sciatic injury and 50% of normal 45 days after irreversible denervation. Wet weight and total protein fell in approximate parallel. Results of assay and electrophoresis (zymograms) showed that muscle esterase was mainly of B type, although A and C type acetylesterases accounted for about 10% of the free or soluble fraction. An increase in the free esterase activity of the homogenate and an absolute increase in A type esterase accompanied denervation and disuse. Bound esterase was 80% released by Triton X-100 and was entirely B type. Zymograms of neonatal muscle were distinguished by the prominence of A-esterases and the presence of an acetylcholinesterase which was not encountered in adults. Over-all, despite some points of similarity, zymograms of neonatal and atrophic adult muscles were distinctly different.

Abnormalities in Brain Esterases in Multiple Sclerosis.

Summary Vertical starch-gel electrophoresis has been used for preparation of esterase zymograms of postmortem specimens of human brain. The specimens derived from 36 cases without a history of significant neurologic disability and 13 cases of multiple sclerosis. Certain esterases hydrolyzing alpha-naphthyl propionate were consistently demonstrable in, and characteristic of, zymograms of white matter from normal specimens. These were absent from zymograms of multiple sclerosis plaques. Alpha-naphthyl acetate zymograms of plaques and macroscopically-normal white matter from multiple sclerosis specimens showed frequently loss of, or diminution in activity of, the 6th, 7th and 9th anodally-migrating esterase bands, but alteration in bands 6 and 7 was not specific for multiple sclerosis. The cholinesterase activity of plaque tissue appeared to be diminished in comparison with that of macroscopically-uninvolved white matter from the same case, and one or more bands were absent in the plaque. The electrophoretic mobility of esterases of the plaque, and sometimes of grossly uninvolved multiple sclerosis white matter, appeared to be greater than that of normal specimens examined under identical conditions of electrophoresis.

Separation of Brain Proteins by Starch Gel Electrophoresis.

Summary Starch-gel electrophoresis has been employed to separate the proteins in human and rat brain. This technic yielded results superior to those attained by paper or agar-gel electrophoresis. Separation of water-soluble proteins on starch-gel yielded 11–12 discrete protein bands in rat brain and 14–15 bands in human white matter. Human white matter proteins are characterized by low concentration of albumin-type proteins.