PROTEIN KINASES IN MYELIN OF RAT BRAIN: SOLUBILIZATION AND CHARACTERIZATION

Myclin from rat brain contained adenosine 3′, 5′-monophosphate (cyclic AMP)-dependent protein kinase activity, which was solubilized by 0.2% Triton X-100 and required exogenous protein substrate for its activity. Also present was a protein kinase which catalysed the phosphorylation of the endogenous substrate and which was neither solubilized by Triton X-100 nor stimulated by cyclic AMP. Sodium fluoride was required to maintain the activity of the endogenous phosphorylation, probably by inhibiting ATPase activity, but had no effect on the phosphorylation of histone by the solubilized enzyme. Protamine and myelin basic protein served as well as histone as a substrate for the solubilized enzyme. A protein kinase modulator had no effect on the endogenous phosphorylation, but inhibited histone phosphorylation by the solubilized enzyme. Cyclic AMP-binding activity was observed in both the solubilized and non-solubilized preparations. The concentration of cyclic AMP required to give half-maximal binding activity of the preparations was about 2.5 nM. The results indicate that the cyclic AMP-binding site of the protein kinase in myelin may partially be accessible, whereas the catalytic site may be integrated into the membrane structure of myelin.     

ACETYLCHOLINESTERASE OF RAT SYMPATHETIC GANGLION: MOLECULAR FORMS, LOCALIZATION AND EFFECTS OF DENERVATION

Abstract— In sucrose gradient centrifugation, acetylcholinesterase (AChE, EC 3.1.1.7.) from the rat superior cervical ganglion (SCG) has been found to contain four molecular forms, characterized by their sedimentation coefficients (4 S, 6.5 S, 10 S and 16 S). Homogenization of the ganglia in various media showed that the 4 S enzyme was readily solubilized in water whereas solubilization of the 6.5 S and 10 S forms was quantitative only in media containing Triton X-100. In order to solubilize the 16 S form, high concentrations of salt (NaCl 1 M) and detergent had to be present. AChE analysed by non-denaturing polyacrylamide gel electrophoresis separated into five bands. Although both distribution patterns were stable, i.e. each form or band preserved its characteristic sedimentation or electrophoretic migration when reanalysed, there was no 1:1 correlation between the forms isolated by sedimentation and the bands obtained by electrophoresis: one band might contain more than one form of enzyme, and conversely one form gave rise to several bands. It was therefore impossible to derive molecular weights from electrophoretic migration in non-denaturing gels. However, it could be shown that the results obtained by both methods of analysis were consistent. Acetylcholinesterase from other nervous structures was analysed: in pre- and postganglionic nerves, the main forms were 10 S and 6.5 S, with a small proportion of 4 S; the 16 S form was not detected. In other sympathetic ganglia, the distribution of forms was identical to that of the superior cervical ganglion. In rachidian ganglia, no 16 S form could be found. Following the section of the preganglionic nerve, the acetylcholinesterase activity of the superior cervical ganglion decreased by 50% in 3 days, and then rose again to about 80% of its original value after 2 weeks. These effects mainly reflected variations in the major 4 S and 10 S forms. The 16 S form, in contrast to its disappearance from denervated muscles, increased transiently during the first 2 weeks after denervation, reaching about twice its original activity. A concomitant cytochemical study of normal and denervated ganglia showed that after preganglionic denervation, AChE localized in the sympathetic neurones decreased markedly and remained low even during the recovery phase. During this period a cholinesterasic activity appeared in the perineuronal glia. Controls established that the enzyme synthetized in the glia is AChE.     

DEVELOPMENT OF ACETYLCHOLINESTERASE MULTIPLE MOLECULAR FORMS IN CHICKEN MUSCLES

Abstract— AChE activity and protein content in chicken ALD and PLD muscles was studied during pre- and postnatal development. Protein content in both muscles increased whereas AChE activity increased in ALD and decreased in PLD during development. All studied values reached the steady-state 3 weeks after hatching.
Electrophoretic separation of the samples showed three molecular forms of AChE present in both adult ALD and PLD muscles. Two molecular forms in ALD muscle increased slowly, one form quickly. On the other hand, the activity of AChE forms in PLD muscle decreased with different rates. It appears from these results that the multiple molecular forms of AChE in muscles are not of the same physiological importance.     

PARTIAL CHARACTERIZATION OF SOLUBLE ACETYLCHOLINESTERASE ISOENZYMES OF THE RAT BRAIN

(1) The 105,000 g supernatant fluid obtained from rat brain was separated by agar-gel electrophoresis. (2) Three isoenzymes, capable of hydrolysing acetylthiocholine, one of them also hydrolysing butyrylthiocholine, were detected. (3) The pH optima and K_m for hydrolysis of acetyl- and butyrylthiocholine by the supernatant fluid were determined. (4) After extraction of acetylcholinesterase isoenzymes from the gel, individual isoenzymes were characterized by pH optima and K_m values. (5) Two of the enzymes were characterized as acetylcholinesterase (EC 3.1.1.7) and one as butyrylcholinesterase (EC 3.1.1.8).     

SUBCELLULAR ANALYSIS OF THE MOLECULAR FORMS OF ACETYLCHOLINESTERASE IN RAT SKELETAL MUSCLE

Acetylcholinesterase (AChE, EC 3.1.1.7) activity of rat gastrocnemius muscle homogenized in 1 M-NaCl and 0.5% Triton X-100 was separated by velocity sedimentation in sucrose gradients into three molecular forms with sedimentation coefficients of about 4S, 10S and 16S. The distribution of homogenate AChE activity among the three peaks was 53, 34 and 13% respectively. The different molecular forms were found to be heterogeneously distributed in subcellular fractions prepared from sucrose homogenates of muscle, as follows: Subfractions of the crude sarcolemmal fraction were prepared by discontinuous sucrose gradient centrifugation. AChE was recovered in the greatest yield and with the highest specific activity in a light density subfraction (0.6/0.8 M-sucrose interface). The AChE activity in this light density subfraction was mainly (81-88%) the 10S form of the enzyme. The velocity sedimentation profiles of the AChE activity in the more dense subfractions were markedly different in that 16S AChE was a major component.     

THE SUBCELLULAR LOCALIZATION OF ACETYLCHOLINESTERASE AND ITS MOLECULAR FORMS IN PIG CEREBRAL CORTEX

Abstract— The distribution of acetylcholinesterase among the subcellular fractions of pig cerebral cortex was determined. The crude mitochondrial and microsomal fractions obtained by differential centrifugation accounted for 75% of the enzyme, with the remainder divided between the crude nuclear and soluble fractions.
The occurrence and distribution of the multiple molecular forms of AChE was the same in all four fractions with the dominant species of molecular weights 350,000, 270,000 and 60,000. Further purification of the mitochondrial fraction by density gradient centrifugation gave a series of membrane fractions with very similar multiple forms. The one possible exception was the fraction containing the purified synaptosomal membranes where one band of mol wt 270,000 predominated, although the other molecular weight entities were present. The electrophoretic pattern of AChE present in the fractionated microsomes was the same as in the crude preparation. The content and pattern of the multiple molecular forms of AChE was therefore the same in all fractions of pig brain, apart from that containing the purified synaptosomal membranes.     

THE RELEASE AND MOLECULAR STATE OF MAMMALIAN BRAIN ACETYLCHOLINESTERASE

Abstract— By incubating the particulate fraction of caudate nucleus from calf brain in ion-free media, about 90 per cent of the AChE activity was brought into solution. The effects of different salts, EDTA and tetracaine on the release were studied. The mol. wt. of the enzyme was determined by gel filtration. About 90 per cent of the activity in a fresh preparation appeared in a form with mol. wt. 80,000. During storage this form was gradually transformed into forms with higher mol. wts. The effects of changes in the ionic environment on the aggregation were investigated. Purification attempts always resulted in the transformation of the enzyme into high mol. wt. forms. If the release was performed in the presence of DEAE-Sephadex-A25, the enzyme no longer aggregated. The cytosol fraction always contained some AChE activity; the significance of the presence of AChE in this fraction is discussed.     

PROPERTIES OF ACETYLCHOLINESTERASE FROM RAT BRAIN

—Acetylcholinesterase (EC 3.1.1.7) from cerebral cortex of mature rats was purified by means of affinity chromatography, to a specific activity of 4.5 mmol acetylthiocholine hydrolysed × min^?1× mg^?1 protein. The enzyme is a glycoprotein and contains a single subunit with a mol. wt of about 80,000. Electrofocusing either a pure or a crude preparation of the enzyme produces six enzymatically active bands with a range of isoelectric points from 5.04 to 5.54. Gel filtration yields oligomers with molecular weights of about 150,000, 320,000, 500,000 and 650,000, with 60 per cent of the activity in the 150,000 fraction. The gel fractions with molecular weights 150,000 and 320,000 produce the same isoelectric patterns. Different subcellular fractions of the cortex show different characteristic isoenzyme patterns.     

大白鼠脑M胆碱受体的溶脱

 本文报告膜蛋白溶脱剂溶脱大鼠脑M胆碱受体的结果,其中0.5％CHAPS,0.35％洋地黄皂苷和10％甘油的混合液效果较好,可溶脱30％的受体,并得到22％有活性的受体。溶脱的受体有较好的稳定性,与膜结合受体有同样的配体结合特异性,可饱和性及可逆性。平衡结合及动力学研究表明溶脱受体和膜结合受体对[~3H]QNB有类似的亲和性。     

大鼠脑中5—羟色胺受体的检定及其在衰老时的变化

采用放射性配基结合分析法,对大鼠大脑皮质的5-HT受体作了检定,并观察了老年大鼠(36月龄)大脑皮质中该受体的变化。证实大鼠大脑皮质存在着丰富的、高亲和力和单一结合位点的5-HT受体。老年大鼠大脑皮质中5-HT受体的数目较成年大鼠(3月龄)明显减少,但亲和力无改变。应用荧光分光技术测定了成年和老年大鼠脑干和大脑皮质5-HT含量,证实老年大鼠上述两个脑区的5-HT含量均有降低。本研究的结果提示,老年大鼠中枢5-HT系统的功能减低,这一变化可能与老年期的一些表现如睡眠障碍、体温低、记忆力减退和易患精神疾病等有关。     

LOCALIZATION, SOLUBILIZATION AND PROPERTIES GANGLIOSIDE TRANSFERASES IN RAT BRAIN OF N-ACETYLGALACTOSAMINYL AND GALACTOSYL GANGLIOSIDE TRANSFERASES IN RAT BRAIN

Abstract— The subcellular distributions of UDP-N-acetylgalactosamine: G_M3 N-acetyl-galactosaminyl transferase and UDP-galactose: G_M2 galactosyl transferase, two enzymes involved in the biosynthesis of gangliosides, were determined in the 7-day-old rat brain by means of synaptosomal fractionation techniques. The enzymes were located on the synaptic membranes and appeared to be closely associated with gangliosides and acetylcholinesterase. Solubilization of the transferase enzymes from the microsomal particles was achieved and differed from the solubilization of acetylcholinesterase and of the total membrane protein. Competition studies suggest that the ^N-acetylgalactosaminyl transferase involved in the formation of G_M2 from G_M3 is different from the N-acetylgalactosaminyl transferase involved in the formation of GalNAoGal-Glc-ceramide from Gal-Glc-ceramide, whereas in contrast, both the formation of G_M1 from G_M2 and of Gal-GalNAc-Gal-Glcceramide from GalNAc-Gal-Glc-ceramide appear to be catalysed by the same galactosyl transferase.     

ONTOGENETIC DEVELOPMENT OF A PHOSPHODIESTERASE ACTIVATOR AND THE MULTIPLE FORMS OF CYCLIC AMP PHOSPHODIESTERASE OF RAT BRAIN

Abstract— The activity of cyclic AMP phosphodiesterase of rat cerebral homogenates increased several-fold between 1 and 60 days of age. Enzyme activity in the cerebellum, on the other hand, did not increase during this period. A kinetic analysis of the phosphodiesterase activity revealed evidence for multiple forms of the enzyme and indicated that the postnatal increase in phosphodiesterase activity of rat cerebrum was due almost exclusively to the high K_m enzyme. In cerebellum, the ratio of the high and low K_m enzyme remained fairly constant during ontogenetic development. Physical separation of the phosphodiesterases contained in 100,000 g soluble supernatant fractions of sonicated brain homogenates by polyacrylamide disc gel electrophoresis confirmed the presence of multiple enzyme forms. In adult rats we found six distinct peaks of phosphodiesterase activity (designated I to VI according to the order in which they were eluted from the column) in cerebellum and 4 forms of the enzyme (Peaks I through IV) in cerebrum. Brains of newborn rats had a different pattern and ratio of phosphodiesterase activities. For example, Peak I phosphodiesterase was undetectable in cerebrum or cerebellum of newborn rats. Moreover, in the cerebellum of newborn rats Peak II was the dominant peak whereas in the cerebellum of adult rats Peak III was the largest peak. A comparison of the multiple forms of phosphodiesterase from the cerebrum of newborn and adult animals suggested that the postnatal increase in phosphodiesterase activity previously seen in crude homogenates was due largely to an increase in a high K, Peak II phosphodiesterase. The ratios of activities of the other peaks and their sensitivities to an activator of phosphodiesterase were similar in newborn and adult rats. An endogenous heat-stable activator of phosphodiesterase was found in cerebrum, cerebellum and brain stem. In newborn rats, the cerebellum contained several-fold less activity of this activator than did cerebrum or brain stem. However, the activity of this activator increased with age in the cerebellum and would appear to have decreased postnatally in cerebrum and brain stem. These results suggest that some multiple forms of phosphodiesterase can develop independently and that changes in activities of these phosphodiesterases may occur by increases in the quantity of enzyme or by changes in the quantity of an endogenous activator of phosphodiesterase.     

THE DETERMINATION OF ACETYLCHOLINESTERASE ACTIVITY OF BRAIN SLICES AND ITS SIGNIFICANCE IN STUDIES OF EXTRACELLULAR ACETYLCHOLINESTERASE

The AChE activity of single slices obtained from the surface of the temporoparietal region of rat brains was measured colorimetrically under anaerobic conditions with acetylthiocholine as substrate. In intact slices from untreated rats AChE activity was only a small proportion of that of homogenates made from the slices, but this proportion increased with the surface area to weight ratio of slices and with an increase in substrate concentration, intact slices not showing substrate inhibition. The inhibition of AChE determined in slices from rats treated with DFP or paraoxon was less than that in homogenates obtained from the slices. When the access of substrate was limited to the cut surface of a slice, the rate of hydrolysis was four times greater than that observed when access was solely from the uncut surface. It is concluded that under anaerobic conditions the substrate diffuses into slices to a depth which is not constant but a function of both substrate and enzyme concentration. Thus the AChE activity of slices cannot be used as a measure of extracellular AChE.     

THE HALF-LIFE OF ACETYLCHOLINESTERASE IN MATURE RAT BRAIN

—The rate of degradation of acetylcholinesterase [EC 3.1.1.7] in mature rat cerebral cortex was determined from the time course of the label introduced into the protein by one intraventricular injection of l -leucine-l-¹⁴C. The half life of the enzyme was 2.84 ± 013 days.     

CHARACTERIZATION OF MULTIPLE FORMS OF BRAIN TUBULIN SUBUNITS

Abstract— Microtubular protein was isolated from rat forebrain by biochemical purification (ammonium sulfate precipitation followed by DEAE cellulose chromatography) or by two cycles of aggregation-disaggregation. The protein subunit structure was examined on two-dimensional electrophoretograms: first dimension, urea isoelectric focusing gel; second dimension, sodium dodecyl sulfate exponential acrylamide slab gel. Two forms of α tubulin were separated in the second dimension on the basis of different rates of migration (α and α₂). Each of these species was further separated into at least three forms with different isoelectric points. β Tubulin was separated into a minor species (BI) and a major species β₂). Multiple subunits were observed using protein from either purification method and in a two-dimensional electrophoretogram of total supernatant proteins from rat brain. Separation and visualization of multiple forms of α and β tubulin is consistent with reports that provide evidence for post-translational modification of these proteins.     

CHARACTERIZATION OF A RAT BRAIN FUCOSYL-TRANSFERASE

Rat brain fucosyl-transferase was solubilized using Triton X–100 detergent, and then purified by electrofocusing. From studies of some exogenous glycoproteinic acceptors, desialylated fetuin appeared to be the most effective substrate. Study of initial velocity patterns gave evidence for a BiBi sequential mechanism, assumed to be a random BiBi with dead end inhibition.     

THE EFFECTS OF SOLUBILIZATION PROCEDURES ON THE RELEASE AND MOLECULAR STATE OF ACETYLCHOLINESTERASE FROM ELECTRIC ORGAN TISSUE

Abstract— A study was made of the effect of various solubilization procedures on the release of AChE from electric organ tissue of the electric eel and on the molecular state of the enzyme. The procedures employed included homogenization in different ionic media or in the presence of detergents, etuymic treatment and chemical modification. Studies were performed on intact electroplax, tissue homogenates and membrane fractions. The apparent AChE activity of intact cells, homogenates and membrane fractions was shown to be governed by diffusion-controlled substrate and hydrogen ion gradients, generated by AChE-catalyscd hydrolysis, leading to a lower substrate concentration and a lower pH in the vicinity of the particulate enzyme.
Treatment of homogenates with NaCl solutions or with NaCl solutions containing the nonionic detergent Triton X-100 causes release of the native'molecular forms of the enzyme (primarily the 18 S species) which aggregate at low ionic strength. For optimal extraction both high ionic strength (e.g. 1 M-NaCl) and the detergent are needed AChE is also solubilized by treatment of tissue homogenates with trypsin, bacterial protease or collagenase. The first two enzymes caused its release as an 11 S non-aggregating form, while collagenase also produces a minor non-aggregating - 16 S component. Treatment of tissue homogenates with maleic anhydride causes release of AChE as a non-aggregating 18 S species. On the basis of the solubilization experiments it is concluded that the interaction of AChE with the excitable membrane is primarily electrostatic. The possible orientation of the enzyme within the synaptic gap is discussed.     

STUDIES ON ACETYLCHOLINESTERASE OF RAT BRAIN SYNAPTOSOMAL PLASMA MEMBRANES

Abstract— A fluorimetric assay has been used to examine some kinetic properties of AChE from synaptosomal plasma membranes prepared from rat brain. The AChE bound to the plasma membranes was compared to that solubilized with Triton X-100 and found to be essentially the same with respect to Michaelis constant and inhibitor constants for several AChE inhibitors. The two forms of the enzyme had slightly different pH optima. The kinetic studies revealed no evidence that synaptosomal plasma membrane AChE has allosteric properties. The solubilized enzyme was further purified by affinity chromatography.     

ISOLATION AND CHARACTERIZATION OF GLUCOCEREBROSIDES FROM ADULT RAT BRAIN1

Abstract— By chromatography on borate-coated silicic acid, glucocerebrosides, galactocerebrosides, sulfatides and sphingomyelins from brain tissue could be efficiently separated. Adult rat brain was found to contain 54.1 ± 1.5 nmol of glucocerebrosides per gram fresh weight. Ninety percent of the glucocere-broside fatty acids were palmitate, stearate and oleate; fatty acids with chain lengths above C₂₀ were virtually absent. No hydroxy fatty acids were found. The long chain bases of adult rat brain glucocerebrosides consisted of 74.6% C₁₈-sphingosine, 24.4% C₁₈-sphinganine and 1.1% C₂₀-sphingosine. These results are compared to those obtained from glucocerebrosides from immature rat brains (Abe & Norton , 1974) and discussed in respect to changes occurring during brain development.     

IDENTIFICATION AND CHARACTERIZATION OF WATER SOLUBLE RAT BRAIN ANTIGENS I. BRAIN AND SPECIES SPECIFICITY

—Some new immunoelectrophoretic methods were applied to the study of brain and species specificity of water-soluble rat brain antigens. Five antigens were brain specific and one, a brain specific antigen, was species restricted.