Kevin D. Barron

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.

Redistribution of rat brain esterases during subcellular fractionation.

Abstract Carboxylic ester hydrolases (nonspecific esterases) and acetylcholinesterase were determined in particulate fractions of rat cerebrum after differential and density-gradient centrifugation. The crude mitochondrial fraction was osmotically shocked by distilled water. When related to the total activity of the original homogenate, 12% of nonspecific esterases and 18.5% of acetylcholinesterase were recovered in the crude mitochondrial fraction. Assays of mitochondrial subfractions showed that 68 and 63.8% of these enzyme activities respectively were recovered in the myelin. The high activity of the myelin subfraction appeared to be due to artifactual redistribution of enzyme. The amount of enzyme released and subsequently bound to subcellular membranes during preparative manipulations might depend on protein-lipid interactions. The synaptic membrane subfraction retained by 0.9 M sucrose differed from the other subfractions in the degree of retention of membrane-bound enzymes during suspension in media of varying pH, osmolality and ionic concentration. These membranes may have a special and physiologically significant molecular organization which differs from that of other membrane fractions.

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.

BRAIN ESTERASES AND PHOSPHATASES IN MULTIPLE SCLEROSIS

There is on record a body of observations, both qualitative (histochemical) and quantitative in nature, which might support the suggestion that phosphatases and esterases are actively involved, however secondarily, in the degradation of myelin lipid which accompanies certain induced central and peripheral neural lesions of experimental animals. l ’ Especially relevant to the postulation of an involvement of esterases in myelin degradation are reports of the experimental production of demyelination by lipolytic enzyme preparations both in uiuo and in vitro.”. Heightened activities of nonspecific phosphatases10-12 and of adenosine triph~sphatase’~ have been described at the margins of plaques of the naturally-occurring human demyelinating disorder, multiple sclerosis, and it would seem possible that phosphatases also may bear some relationship to the demyelinating process. In contrast to the observations cited are data which might indicate a role of some hydrolytic enzymes, such as 5-n~cleotidase’~. l 5 and butyrylcholinesterase’8, in myelin synthesis and maintenance. Thus, there is a striking topographical parallelism between the appearance of 5-nucleotidase activity in developing (myelinating) rat brain and the deposition of myelin.“ Cholinesterase and 5-nucleotidase activities are diminished in the plaques of multiple s c l e r~s i s . ’~ -~~ The reports cited above would support the relevance of a study of hydrolytic enzymes, especially phosphatases and esterases, in tissue from the most prevalent of human demyelinating diseases, namely, multiple sclerosis. During the past several years we have investigated these enzymes in human brains affected by multiple sclerosis and the possibly allied disorder, Schilder’s disease. Data derived from the pathologic material have been compared to findings obtained from normal specimens. In view of the demonstration in the recent past that many hydrolases occur in multiple molecular forms’8 (“isozymes”ly~ z o ) our work has employed starch gel electrophoresis and the zymogram’8 technique in addition to other methods. A part of our data has been reported p r e v i o ~ s l y . ~ ~ ~ 17. zl, ** This paper will describe new observations and will summarize the findings to date.

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.

MULTIPLE MOLECULAR FORMS OF BRAIN HYDROLASES.

Publisher Summary This chapter discusses multiple molecular forms of brain hydrolases. The various hydrolases are separated by vertical starch-gel electrophoresis, and visualization of the zones of enzymatic activity is accomplished by applying various histochemical techniques for several enzymes to the gel, for nonspecific esterases. The enzymes studied include nonspecific esterases, acetyl- and butyrylcholinesterases, phosphomonoesterases, and thioesterase. Organophosphorus inhibitors and other compounds are used to characterize the enzymes in terms of isozyme homogeneity, and densitometric and other quantitative techniques are used to evaluate the degree of sensitivity of these isozymes to the inhibitors. Data are obtained on normal and pathological human material as well as animals, and some developmental and experimental studies are reported. Alterations in isozyme patterns are shown to occur during brain development, in the muscle esterases after denervation, and in human pathological tissue. Cytochemical studies have indicated a lysosomal localization of the nonspecific esterase, which are then adsorbed on microsomes in the process of preparation. A lysosomal locus provides presumptive evidence of a hydrolytic function of these esterases because the lysosome are shown to contain only hydrolytic enzymes to date.