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 CARBAMYLCHOLINE-STIMULATED PHOSPHATIDYL INOSITOL TURNOVER IN RAT CEREBRAL CORTEX IN VIVO

Abstract— The intraventricular injection of 40 μCi of ³²P_i (carrier free) into adult rats resulted in maximum incorporation of ³²P_i into the phosphatidyl inositol of the whole cortex after 20 h. A further intraventricular injection of 2 nmol carbamylcholine plus 0.02 nmol eserine resulted in a 23% decrease in the specific activity of phosphatidyl inositol after 20 min. The specific radioactivities of phosphatidyl choline, phosphatidyl ethanolarmine and phosphatidyl serine were not changed. Cerebral cortex from rats treated in this way was subjected to an extensive subcellular fractionation. It was found that the specific radioactivity of the phosphatidyl inositol of the synaptic vesicle fraction showed a reduction of 60%. No other fractions showed effects of this magnitude.     

LOCALIZATION AND MULTIPLE FORMS OF ACETYLCHOLINESTERASE IN SEA URCHIN EMBRYOS

Acetylcholinesterase during the development of the sea urchin Pseudocentrotus depressus was examined by enzyme assay (the colorimetric method of E llman et al. ), histochemistry (a Cu-thiocholine method), polyacrylamide gel electrophoresis and DEAE-Sephadex ion exchange chromatography.
The enzyme activity is detected in the unfertilized egg, remains low during cleavage, elevates slightly through gastrulation, and then increases rapidly thereafter. The intense activity is localized in the mesenchyme cells associated with the larval skeleton of young pluteus larvae, and their cell membranes and nuclear envelops. Soluble enzyme accounts for 60% of the total activity. The additional 34% is extracted by 1% Triton X-100 from particulates. The soluble enzyme consists of two forms. Both are strongly acidic proteins which are similar in electric charge, but dissimilar in size, being 180,000 and 280,000 in molecular weights. The enzyme released from the membrane by the detergent possesses a component which is not present in the soluble complement of the enzyme. It is not a secondary product of the soluble enzyme interacting with the detergent.
Acetylcholinesterase serves as a marker of late differentiation and regional differentiation in the sea urchin embryo.     

SUBCELLULAR DISTRIBUTION OF NORADRENALINE IN GUINEA-PIG CEREBRAL CORTEX AND SPLEEN

Abstract— The distribution of noradrenaline (NA) in subcellular fractions of guinea-pig cerebral cortex and spleen was determined by differential and density gradient centrifugation. Of the primary fractions, the microsomal fraction from both tissues was enriched in NA, that of the spleen having the higher specific activity. Microsomal fractions were therefore placed on gradients and NA determined in the subfractions since these fractions appeared suitable preparations in which to search for discrete populations of vesicles. So that the non-occluded micro-particulate bound noradrenaline (MPBNA) content of gradient subfractions could be measured, [³H]NA was used to control for the diffusion and or adsorption of free NA, and occluded lactate dehydrogenase was used to estimate the amount of entrapped MPBNA and soluble NA. Non-occluded MPBNA on gradients from microsomal fractions of cerebral cortex formed a single peak mainly in subfraction F (0.6-0.8 m -sucrose). Spleen microsomal fractions, however yielded two peaks of MPBNA. one in sub-fractions D to G (0.4-1.0 m -sucrose) and the other in sub-fraction J (1.4 m -sucrosc); electron microscopy showed that the latter subfraction contained large vesicles.
Since there were unexpectedly small amounts of MPBNA in microsomal subfractions D and E of cerebral cortex, the synaptosome fraction was investigated. Following water treatment of synaptosomes. MPBNA formed a peak in subfraction E (0.4-0.6 m -sucrose) with smaller amounts in subfractions D and F (0.4 and 0.6 0.8 m -sucrose).     

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.     

SOLUBILIZATION AND PHYSICOCHEMICAL CHARACTERIZATION OF RAT BRAIN ACETYLCHOLINESTERASE: DEVELOPMENT AND MATURATION OF ITS MOLECULAR FORMS

Abstract— We have solubilized two active molecular forms of AChE from rat brain and compared them to the molecular forms solubilized from rat muscle. One of these forms, in muscle, as well as in brain, is easy to solubilize without detergent (ES form–apparent sedimentation coefficient without detergent: 4.6s); the other is hard to solubilize and we obtained a nearly total solubilization only in the presence of detergent (HS form–apparent sedimentation coefficient in presence of detergent: 10.3s). These two molecular forms are glycoprotein in nature. They interact with detergent (Triton X-100), as demonstrated by a comparison of their hydrodynamic parameters (determined by sucrose gradient centrifugation and molecular filtration) in the presence and absence of detergent. In the absence of detergent, their molecular weights are 115,000 for the ES form and 435,000 for the HS form. We did not find the molecular form in brain which seems to be specific to the muscle endplate region. at any stage of its development (EP form–solubilized by detergent–apparent s value in presence of detergent: 16.2s).
During development or maturation of the rat brain, the relative proportion of the HS form to the ES form increases; its absolute amount also increases (by more than a factor of 7 during the first month after birth). The ES form seems to be established at its adult level at the time of birth, before the large increase in the HS form. The proportion of each form in the adult rat brain remains constant: 90% of the total activity is represented by the HS form.     

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.     

IDENTIFICATION OF MULTIPLE FORMS OF AMINOBUTYRATE TRANSAMINASE IN MOUSE AND RAT BRAIN : SUBCELLULAR LOCALIZATION

—(1) At least four distinct molecular forms of 4-aminobutyrate: 2-oxoglutarate aminotransferase from mouse and rat brain, have been separated by electrophoresis on paper, cellogel, agargel, silicagel and by immunoelectrophoresis. (2) The existence of specific typical electrophoretic profiles in mitochondrial and extramitochondrial compartments was shown. (3) A differential effect of pH on the anionic and cationic 4-aminobutyrate:2-oxoglutarate aminotransferase transaminase activities has been shown. (4) The possible consequences of the 4-aminobutyrate: 2-oxoglutarate aminotransferase isozyme compartimentation on the local availability of γ-aminobutyric acid pools has been discussed.     

CHARACTERIZATION AND LOCALIZATION OF ACID HYDROLASE ACTIVITY IN THE SYNAPTOSOMAL FRACTION FROM RAT CEREBRAL CORTEX

Synaptosomes were prepared from the cerebral cortex of adult rats by a rapid technique of centrifugation in a Ficoll-sucrose discontinuous gradient. The synaptosomal fraction contained 40 per cent of the total gradient activity of acid α-naphthyl phosphatase (EC 3.1.3.2). Quantitative electron microscopy of this fraction revealed rare, typical, extrasynaptosomal dense body lysosomes. pH-activity profiles of free and Triton X-100 (total) activities were prepared for α-naphthyl phosphatase, β-glucuronidase (EC 3.2.1.31), β-galactosidase (EC 3.2.1.23), arylsulfatase (EC 3.1.6.1) and N-acetylglucosaminidase (EC 3.2.1.30). The ratios of total to free activity varied in the order: arylsulfatase > β-galactosidase > β-glucuronidase > N-acetylglucosaminidase > acid phosphohydrolase. Incubation of synaptosomal fractions at pH 5 and 37°C produced significant activation of β-galactosidase and N-acetylglucosaminidase but no activation of cryptic lactate dehydrogenase (EC 1.1.1.27). Hyposmotic suspension and subfractionation of the synaptosomal fraction produced considerable solubilization of lactate dehydrogenase, arylsulfatase and β-galactosidase but only partial liberation of α-naphthyl phosphatase, the remainder being associated with synaptosomal membrane fragments. Incomplete equilibrium sedimentation of synaptosomes in a continuous sucrose gradient (0·55-1·5 M) provided a broad lactate dehydrogenase and Na + K ATPase (EC 3.6.1.4) peak (peak I) at low sucrose densities. β-Glucuronidase, β-glucosidase and α-naphthyl phosphatase were significantly present in peak I. Conversely, N-acetylglucosaminidase, arylsulphatase and β-galactosidase were predominantly located in denser particles sedimenting through 1·2 M sucrose (peak II). Electron microscopy confirmed the heterogeneity of this second peak and the presence of numerous extrasynapto-somal dense body lysosomes.     

SUBCELLULAR LOCALIZATION OF NEWLY-FORMED [3H]ACETYLCHOLINE IN RAT CEREBRAL CORTEX IN VITRO

—Slices from rat brain cortex were incubated for either 5 or 60 min in a medium containing [³H]choline and 4·7 or 25 mm -KCl. Bioassayable ACh and labelled ACh were determined in the incubation medium, in the total tissue homogenate and in subcellular fractions. Raising the KCl concentration from 4·7 to 25 mm stimulated the release and synthesis of total and of labelled ACh. In medium containing 25 mm -KCl the amounts of ACh decreased in the tissue and in the nerve ending cytoplasm, but remained constant in the synaptic vesicles. After incubation in 25 mm -KCl medium the ACh in the vesicles was labelled to the same extent as the cytoplasmic ACh but after incubation in 4·7 mm -KCl medium vesicular ACh was labelled less than cytoplasmic ACh. During 5 min incubation in medium containing 25 mm -KCl the ratio of labelled to total ACh was much higher in the medium than in the homogenate, the vesicles or the cytoplasm. During the last 15 min of the 60 min incubation the ratio of labelled to total ACh in the medium was still higher than that in the tissue fractions, but less so than during the 5 min incubation. It is concluded that the vesicular and cytoplasmic fractions are not identical with the store in the tissue from which newly-synthesized ACh is preferentially released.     

PURIFICATION AND CHARACTERISTICS OF RNase INHIBITOR FROM PIG CEREBRAL CORTEX

Abstract— An RNase inhibitor has been purified from pig cerebral cortex by DEAE-cellulose and hydroxylapatite chromatography and Sephadex G-100 gel filtration. The purified RNase inhibitor could be resolved into a major band (about 80–85 per cent of total protein) and several minor components by polyacrylamide gel electrophoresis.
The ultraviolet absorption curve of the purified RNase inhibitor indicated a typical protein spectrum. The inhibitor was inactivated by digestion with trypsin or prozyme, and by heating at 70ºC for 5 min. The inhibitor was also inactivated by an SH reagent such as p -chloromercuribenzoate. The inhibitor did not affect RNase T₁. It has been suggested that the inhibitor is an acidic protein and also a SH-protein. The molecular weight of the RNase inhibitor was estimated to be about 60,000.     

SUBCELLULAR LOCALIZATION OF PROTEIN CARBOXYL-METHYLASE AND ITS SUBSTRATES IN RAT PITUITARY LOBES

Subcellular distribution of protein carboxyl-methylase (PCM) and its endogenous substrates the methyl acceptor proteins (MAP) have been studied in the anterior, the posterior and the intermediate lobes of the rat pituitary gland. In all three lobes, PCM was found to be a cytosolic enzyme whereas the highest MAP specific capacity was observed in the lysate of the granular fractions. The methylated proteins from the lysates of the granular fractions and the cytosolic fractions were analyzed with a novel electrophoretic system which prevents the hydrolysis of the protein-methyl esters. Gel electrophoretic profiles from the lysates of the granular fractions were different from those of the cytosolic fractions. In the lysatcs of the granular fractions. the MAP had a molecular weight of less than 25,000 suggesting that the methylated polypeptides in these fractions were the pituitary peptide hormones (and their subunits) and neurophysins.     

MEMBRANE AFFINITIES AND SUBCELLULAR DISTRIBUTION OF THE DIFFERENT MOLECULAR FORMS OF CHOLINE ACETYLTRANSFERASE FROM RAT

Choline acetyltransferase from rat brain is present in three different molecular forms with isoelectric points at pH 7·4-7.6, 7·7-7·9 and 8·3. The three forms were identified in a highly purified enzyme preparation, in a preparation of synaptosomes and in a cyto-plasmic preparation from disrupted axons and perikarya (fraction S₃). The three molecular forms differed in their affinities for synaptosome membranes and for a cation exchange resin (CM-Sephadex C-50). The positive surface charges of the different molecular forms and their affinities for membranes correlated well with their isoelectric points. The molecular form with jsoelectric point 8·3 had the largest positive surface charge and the highest membrane affinity. On isoelectric focusing of an extract from rat brain synaptosomes, the molecular form with isoelectric point 8·3 formed a complex with a negatively charged compound, presumably a protein. A method was developed to remove this compound by treatment with DEAE-Sephadex or by precipitation with vinblastine. These procedures are similar to methods known to remove the neurotubular protein. The complex formation did not occur in fraction S₃.     

DISC ELECTROPHORETIC SEPARATION OF PROTEINS IN NEURONAL, GLIAL AND SUBCELLULAR FRACTIONS FROM CEREBRAL CORTEX

Abstract— Soluble proteins were studied in preparations from rabbit brain cortex enriched in neuronal or glial cells and in subcellular cortical fractions. Analytical polyacrylamide gels were used for acidic (pH 9-5) and basic (pH 4-3) proteins and qualitative and quantitative differences are described. The isozymes of lactic dehydrogenase, brain specific proteins and radioactive labelling patterns were used to characterize some soluble proteins.     

UPTAKE AND RELEASE OF TAURINE FROM CEREBRAL CORTEX SLICES AND THEIR SUBCELLULAR COMPARTMENTS

—Cortex slices, synaptosomes and C-6 glioma cells were used to study [³⁵S]taurine uptake and its electrically-stimulated release. After exposure to taurine at two concentrations, the synaptosome preparation subsequently derived from the slices contained 41% of the particle-bound taurine and 16% of the total in the tissue. The uptake of [¹⁴C]GABA by C-6 glioma cells was inhibited 3-fold more by β-alanine than by l -DABA, whilst synaptosome preparations showed the opposite pattern, l -DABA being 2 or 3 times more effective than β-alanine. [³⁵S]Taurine uptake inhibition by l -DABA was low for synaptosomes and C-6 glioma, whereas β-alanine showed considerable effect on C-6 glioma (41%) and slices of white matter (ependyma; 50%). Synaptosome preparations showed little effect with β-alanine. When 30 min rather than 5 min incubations were employed, β-alanine depressed [³⁵S]taurine uptake by cortex slices by 30%. Taurine was taken up by a calcium-dependent mechanism and subcellular fractionation indicated that the synaptosome fraction showed losses commensurate with the net taurine release when low stimulation currents were used.     

SPECIFICITY AND KINETIC PROPERTIES OF MONOSACCHARIDE UPTAKE INTO GUINEA PIG CEREBRAL CORTEX IN VITRO

Abstract— The uptake into the non-raffinose space of cerebral cortex slices of a number of ¹⁴C-labelled glucose analogues has been studied. Evidence on competition with glucose for the transport process has been used to derive information on the substrate specificity of sugar uptake to the brain. The kinetic properties of the uptake of 2-deoxygIucose indicate that the transport is a facilitated process rather than diffusion. Classical competition between glucose and 2-deoxyglucose for transport is shown and arguments are advanced for regarding glucose as a competitive inhibitor of 2-deoxyglucose transport. The apparent K_m for deoxyglucose is 10 mM and for glucose is suggested to be of the order of 5 mm , The value of such a kinetic approach to sugar transport in various conditions is discussed.     

SUBCELLULAR DISTRIBUTION OF PYRUVATE KINASE (EC 2.7.1.40) IN CEREBRAL CORTEX

—The subcellular distribution of pyruvate kinase (EC 2.7.1.40) in the cerebral cortex of the rat was studied. The enzyme, which had been previously reported in the cytoplasm, was found to be present in synaptosomal, microsomal and mitochondrial fractions as well. The activity of the enzyme in the synaptosomal fraction was localized predominantly in the synaptosomal membrane and was not dissociated by repeated washing or recentri-fuging in a sucrose gradient. Some kinetic parameters of the membrane-associated pyruvate kinase were measured.     

TURNOVER OF PROTEIN PHOSPHORUS IN RESPIRING SLICES OF GUINEA PIG CEREBRAL CORTEX: CELLULAR LOCALIZATION OF PHOSPHOPROTEIN SENSITIVE TO ELECTRICAL STIMULATION

Abstract— In experiments designed to localize the increased turnover of phosphoprotein-P which occurs in respiring brain slices as a result of electrical stimulation, a cell separation procedure was used to prepare a fraction enriched in neuronal cell bodies from incubated slices labelled with [³²P]phosphate. Labelled phosphoprotein was found to be twice as concentrated in the neuron-enriched fraction as in other fractions. Electrical stimulation for 10 s increased the rate of incorporation of [³²P]phosphate into phosphoproteins in the neuron-enriched fraction by 25 per cent ( P < 0.05), but had no effect on incorporation into a partially purified glial fraction contaminated with neuropil and cell debris.     

THE DISTRIBUTION OF SOLUBLE AND MEMBRANE-BOUND FORMS OF GLUTAMINASE IN PIG BRAIN

Abstract— About 10% of the glutaminase activity associated with pig brain mitochondria was readily extractable by a variety of techniques but the remainder was very resistant to extraction. These two forms, which have been termed the soluble and membrane-bound forms respectively, have been shown to differ in their responses to activation by phosphate and phosphate-borate containing buffers. Submitochondrial fractionation studies indicated that the soluble form was located in the mitochondrial inner matrix whereas the membrane-bound form was associated with the inner membrane. The mitochondria associated with the synaptosomes were found to contain only the membrane-bound form of the enzyme whereas both forms were present in the free brain mitochondria.