Preparation of chick brain synaptosomes and synaptosomal membranes

A method is described for the preparation of synaptosomes and synaptosomal membranes from chicken brain. Procedures for isolating rat synaptosomal membranes could not be used directly; several modifications of existing procedures are reported. Purity of the subcellular and subsynaptosomal fractions was monitored by electron microscopy and measurements of ferrocytochrome $\text{c}$: oxygen oxidoreductase (EC 1.9.3.1.), monoamine: oxygen oxidoreductase (deaminating) (EC 1.4.3.4), rotenoneinsensitive NADH: cytochrome $\text{c}$ oxidoreductase (EC 1.6.99.3), NADPH: cytochrome $\text{c}$ oxidoreductase (EC 1.6.99.1), orthophosphoric monoester phosphohydrolase (EC 3.1.3.2), ATP phosphohydrolase (EC 3.6.1.4), and levels of RNA. Microsomes are the main contaminant of the synaptosomal membrane fraction. Mitochondrial and lysosomal enzymes occur in lesser amounts. No myelin contamination was observed. Marker enzymes for contaminants suggest that these synaptosomal membranes are as pure as membranes described by others, and the specific activity of a neuronal membrane marker, $\text{(}\text{Na}{}^{\mathrm{+}}\text{?}\text{K}{}^{\mathrm{+}}\text{)-}\text{activated}$ ATPase, is as high as other preparations. Levels of this enzyme in the membrane fraction are enriched 13-fold over homogenate ATPase levels.     

Development of a Membrane-Bound Respiratory System Prior to and During Sporulation in Bacillus cereus and Its Relationship to Membrane Structure

Bulk membrane fragments were prepared from cells of Bacillus cereus ATCC 4342 harvested at different stages of growth and sporulation and examined for enzymes involved in electron transport functions. The presence of succinate: DCPIP oxidoreductase (EC 1.3.99.1), succinate: cytochrome c oxidoreductase (EC 1.3.2.1), NADH:DCPIP oxidoreductase (EC 1.6.99.1), NADH:cytochrome c oxidoreductase (EC 1.6.2.1), succinate oxidase [succinate: (O(2)) oxidoreductase, EC 1.3.3.1], and NADH oxidase [NADH:(O(2)) oxidoreductase, EC 1.6.3.1] were demonstrated in membrane fragments from vegetative cells, early and late stationary-phase cells, and in cells undergoing sporulation. During the transition from a vegetative cell to a spore, there was a significant increase in the levels of enzymes associated with energy production via the electron transport system. Cytochromes of the a, b, and c type were detected in all membrane preparations; however, there was a marked increase in the level of cytochromes by the end of vegetative growth which remained throughout sporulation; there were no qualitative changes in the cytochromes throughout growth and sporulation. Sporulation was inhibited by cyanide, stressing the significance of the electron transport system. Enzyme activities were partially masked in washed membrane fragments; however, unmasking (stimulation) was achieved by sodium deoxycholate, sodium dodecyl sulfate, or Triton X-100. The degree of enzyme masking was less in vegetative cell membrane fragments than in membranes prepared from stationary-phase or sporulating cells. Results indicate the development of a membrane-bound electron transport system in B. cereus by the end of growth and prior to sporulation, which results in an increased masking of a number of enzymes associated with the terminal respiratory system of the cell.     

Characterization of an ATP diphosphohydrolase (EC 3.6.1.5) in synaptosomes from cerebral cortex of adult rats

Data from the literature have demonstrated that synaptosomal preparations from various sources can hydrolyze externally added ATP. Various authors characterized this activity as an ecto-ATPase. In the present report, we demonstrate that synaptosomal preparations obtained from the cerebral cortex of rats show ATPase activity that could not be dissociated from ADPase activity, suggesting that an ATP-diphosphohydrolase is involved in ATP and ADP hydrolysis. Furthermore, the ATP and ADP hydrolysis could not be attributed to associations of enzymes that could mimic an ATP-diphosphohydrolase because none of the following activities were detected in our assay conditions inorganic pyrophosphatase, adenylate kinase, or nonspecific phosphatases. A possible association between an ATPase and an ADPase was excluded on the basis of both the kinetics and much additional data on inhibitors, ion dependence, pH, etc. The present results demonstrate that in synaptosomal preparations from cerebral cortex an ATP-diphosphohydrolase is involved, at least in part, in ATP and ADP hydrolysis.Abbreviations DCCD dicyclohexylcarboiimide - EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Pi inorganic phosphate Enzymes ATP diphosphohydrolase, Apyrase (EC 3.6.1.5) - ATPase ATP phosphohydrolase (EC 3.6.1.3) 5-nucleotidase (EC 3.1.3.5) Hexokinase (EC 2.7.1.1) Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) Adenylate kinase (EC 2.7.4.3) Inorganic pyrophosphatase (EC 3.6.1.1) - ATP pyrophosphohydrolase (EC 3.6.1.8) - LDH lactate dehydrogenase (EC 1.1.1.27) - SDH succinate dehydrogenase (EC 1.3.1.6) - ACHE acethylcholinesterase (EC 3.1.1.7) - G-6-Pase glucose-6-phosphatase (EC 3.1.3.9) - NADPH cytoehrome c oxidoreductase (NCR) (EC 1.6.2.4)     

Subcellular distribution of marker enzymes and of neurotoxic esterase in adult hen brain.

Neurotoxic esterase (NTE) is now regarded as the site of the primary biochemical lesion in the delayed neuronal degeneration produced by certain organophosphorus esters. Since hens are the species of choice in studies of this neuropathy the subcellular distribution of NTE and marker enzymes in adult hen brain was carried out. Up to 70%, of NTE was recovered in a microsomal fraction (P₃) which was also enriched in 5′-nucleotidase (5′-ribonucleotide phosphohydrolase EC 3.1.3.5), a plasma membrane marker. The protein content of this fraction (31% of the parent homogenate) is double that of equivalent mammalian brain fractions. The LDH distribution suggests that the P₃ fraction contained many small synaptosomes. Subfractionation of microsomes by rate and equilibrium centrifugation on sucrose density gradients segregated the RNA but failed to separate the NTE. 5′-nucleotidase and glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase EC 3.1.3.9) from each other. NTE was considerably concentrated (2–5 times) in subfractions of the P₂ fraction, which are believed to be enriched in synaptosomal membranes. A similar localization of NTE and AChE was found in subfractions of P2 from neonatal chick brain. Axon fragments contained a significant amount of NTE which was not associated with the myelin. Nuclear and mitochondrial fractions were low in NTE. Microsomes could be partitioned in biphasic aqueous polymer systems, but with little enrichment of NTE. The possible association of NTE with synaptosomal membranes suggests that early events in organophosphorus neuropathy may occur at the axonal (? synaptic) surface.     

Occurrence of Sialyltransferase Activity in the Synaptosomal Membranes Prepared from Calf Brain Cortex

The possible occurrence of sialyltransferase activity in the plasma membranes surrounding nerve endings (synaptosomal membranes) was studied, using calf brain cortex. The synaptosomal membranes were prepared by an improved procedure which provided: (a) a ?nerve ending fraction” consisting of at least 85% well-preserved nerve endings and containing only small quantities of membranes of intracellular origin; (b) a ?synaptosomal membrane fraction” carrying high amounts of authentic plasma membrane markers (Na⁺-K⁺ ATPase, 5′-nucleotidase, sialidase, gangliosides) with values of specific activity four to fivefold higher than those in the ?nerve ending fraction” and very small amounts of cerebroside sulphotransferase, marker of the Golgi apparatus, and of other markers of intracellular membranes (rotenone-insensitive NADH and NADPH: cytochrome c reductases), the specific activities of which were, respectively, 0.5- and 0.7-fold that in the ?nerve ending fraction”. Thus the preparation of synaptosomal membranes used had the characteristics of plasma membranes and carried a negligible contamination of membranes of intracellular origin. The distribution of sialyltransferase activity in the main brain subcellular fractions (microsomes; P₂ fraction; nerve ending fraction; mitochondria) resembled most closely that of thiamine pyrophosphatase, the enzyme known to be linked to the Golgi apparatus and the plasma membranes and of acetylcholine esterase, the enzyme known to be linked to either intracellular or plasma membranes. The enrichment of sialyltransferase activity in the ?synaptosomal membrane fraction”, referred to the ?nerve ending fraction”, was practically the same as that exhibited by authentic plasma membrane markers. All this is consistent with the hypothesis that in calf brain cortex sialyltransferase has two different subcellular locations: one at the level of intracellular structures, most likely the Golgi apparatus (as described by other authors), the other in the synaptosomal plasma membranes. The basic properties (pH optimum, V/S, V/t and V/protein relationships) and detergent requirements of the synaptosomal membrane-bound sialyltransferase were established. The highest enzyme activities were recorded on exogenous acceptors, lactosylceramide and ds -fetuin. The K_m values for CMP-NeuNAc were different using lactosylceramide and ds -fetuin as acceptor substrates (0.57 and 0.135 mm , respectively); the thermal stability of the enzyme acting on glycolipid acceptor was higher than that on the glycoprotein acceptor; the effect of detergents was different when using glycoprotein from glycolipid acceptors; no competition was observed between lactosylceramide and ds -fetuin. Thus the synaptosomal membranes carry at least two different sialyltransferase activities: one acting on lactosylceramide (and glycolipid acceptors), the other working on ds -fetuin (and glycoprotein acceptors). Ganglioside GM₃ was recognized as the product of synaptosomal membrane-bound sialyltransferase activity working on lactosylceramide as acceptor substrate.     

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.     

Measurement of proton pump activity of the thermophilic bacterium PS3 and Nitrobacter agilis at the cytochrome oxidase level using total membrane and heptyl thioglucoside

It is possible to prepare liposomal vesicles by solubilization of total bacterial membranes with n-heptyl beta-D-thioglucoside followed by reconstitution into proteoliposomes by a freeze-thaw-sonication procedure with soybean phospholipids. The resulting proteoliposomes from total membrane fraction of sufficiently aerated cells of the thermophilic bacterium PS3 containing cytochrome aa3 showed a reasonable H+ pumping activity upon addition of reduced cytochrome c. On the other hand, the proteoliposomes reconstituted from air-limited PS3 cells containing cytochrome o and those from Nitrobacter agilis cells containing cytochrome aa3 did not show H+ pumping upon addition of reduced cytochrome c, although the vesicles showed "respiratory control"; 3-4-fold stimulation of oxygen consumption took place upon addition of an uncoupler. In proteoliposomes prepared from PS3 membranes by this method, H+-translocating ATPase (F0 X F1) was successfully reconstituted as well, suggesting that this method has wide applicability for investigation of enzymes catalyzing transmembrane processes.     

The localization of the anion-sensitive ATPase activity in corneal endothelium

The localization of the anion-sensitive ATPase (EC 3.6.1.3) of bovine corneal endothelium has been investigated. Homogenates were fractionated by differential and density gradient centrifugation, into fractions enriched in plasma membranes and mitochondria. (Na+ + K+)-ATPase (EC 3.6.1.3) and cytochrome oxidase (EC 1.9.3.1) were used as marker enzymes for these two cell components, and glucose-6-phosphatase (EC 3.1.3.5) was used to identify endoplasmic reticulum. 5'-Nucleotidase (EC 3.1.3.5) was also measured but was found not to be exclusively associated with any one cell component. The activity of the anion-sensitive ATPase (HCO3--ATPase) was measured in suspensions that were frozen and thawed before assay in order to expose latent enzyme activity. The fraction containing the greatest amount of (Na+ + K+)-ATPase (35%) contained only 6% of the cytochrome oxidase and HCO3--ATPase. Conversely, the mitochondrial fraction, containing 40% of the cytochrome oxidase, contained about 40% of the HCO3--ATPase, but only 7% of the (Na+ + K+)-ATPase. The recoveries and relative degree of purification of the cytochrome oxidase and HCO3--ATPase were also nearly identical in the other fractions examined. It was concluded that the anion-sensitive ATPase activity of the corneal endothelium is located solely in the mitochondria and not in the plasma membrane. Consequently, any role that the enzymes may have in the transport of bicarbonate across this tissue, which had been suggested in earlier studies, must be an indirect one.     

The isolation and characterization of the plasma membrane of cultured chinese hamster ovary cells.

Plasma membranes were prepared from cultured Chinese hamster ovary cells utilizing a two-phase polymer system and were characterized by enzymatic and chemical assay, and by electron microscopy. The usual degree of purification of presumptive membrane markers such as Na+-K+ ATPase (ATP phosphohydrolase, EC 3.6.1.3) ranged from three-to eightfold. Gel electrophoresis in SDS revealed several polypeptides and two glycopeptides which were enriched in the plasma membrane fraction.     

Actomyosin-like ATPase extracted from plain synaptic vesicle fractions

Ca²⁺-sensitive Mg²⁺-dependent ATP phosphohydrolase (EC 3.6.1.3, ATPase) was extracted from the plain synaptic vesicle fractions that were virtually devoid of contamination. The protein pattern of the ATPase preparation on SDS polyacrylamide gel electrophoresis closely resembled that of actomyosin from skeletal muscle. The finding suggests that the main components of the ATPase are actin- and myosin-like proteins of the brain (stenin and neurin). Microsome and synaptosomal plasmalemma fractions were extracted under the same conditions to examine the possibility that the ATPase extracted derived from contaminating particulates. An entirely different ATPase was extracted from microsomes, and no protein from plasma membranes. Although Ca²⁺-sensitive Mg²⁺-dependent ATPase was extracted from coated vesicle fraction, the electrophoretic pattern was dissimilar to that of the ATPase from plain synaptic vesicle fractions. It may be inferred that the whole complex of neurostenin is located in plain synaptic vesicles from the brain.     

Synthesis of the mitochondrial inner membrane in cultured Xenopus laevis oocytes.

The purpose of this study was to investigate the contribution of mitochondrial and cytoplasmic protein synthesis to the biogenesis of cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase EC 1.9.3.1) and rutamycin-sensitive adenosine triphosphatase (ATP phosphohydrolase EC 3.6.1.3) in cultured oocytes of the toad, Xenopus laevis. X. laevis cytochrome oxidase was purified over 23-fold with respect to specific activity and over 29-fold with respect to specific heme a content from oocyte submitochondrial particles. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate separated the enzyme into six subunits with molecular weights of 44,000, 33,000, 23,000, 17,000, 12,000 and 9,500. the synthesis of the three larger subunits is sensitive to chloramphenicol (an inhibitor of mitochondrial protein synthesis), indicating that these subunits are made on mitochondrial ribosomes; the synthesis of the three smaller subunits is sensitive to cycloheximide (an inhibitor of cytoplasmic protein synthesis) and therefore occurs on cytoplasmic ribosomes. X. laevis rutamycin-sensitive ATPase, purified over 19-fold from oocyte submitochondrial pparticles, consists of 10 subunits with molecular weights of 56,000, 53,000, 41,000, 32,000, 29,000, 24,000, 21,000, 17,500 (2), and 11,500 on sodium dodecyl sulfate-polyacrylamide gels. The 29,000, 21,000, and one of the 17,500-dalton polypeptides are synthesized in the presence of cycloheximide and are, therefore, products of mitochondrial protein synthesis; the synthesis of the remaining seven subunits occurs in the presence of chloramphenicol, indicating that these subunits are made on cytoplasmic ribosomes. The synthesis of protein by mitochondria in cultured oocytes appears to be dependent upon cytoplasmic protein synthesis. In the presence of cycloheximide, the mitoribosomal synthesis of the subunits of cytochrome oxidase and rutamycin-sensitive ATPase is detectable only after a prior inhibition of mitochondrial protein synthesis by chloramphenicol. Oocyte mitochondrial ribosomes synthesize at least nine polypeptides after chloramphenicol treatment, three of which are components of neither cytochrome oxidase nor rutamycin-sensitive ATPase.     

Plasmalemma from the roots of cucumber: Isolation by two-phase partitioning and characterization

Plasmalemma was isolated from the roots of 2-week-old cucumber plants ( Cucumis sativus L. cv. Rhensk druv) by utilizing an aqueous polymer two-phase system with 6.5%:6.5% (w/w) Dextran T500 and polyethylene glycol (PEG) 3350 at pH 7.8. The plasmalemma fraction comprised ca 6% of the membrane proteins contained in the microsomal fraction. The specific activity of the plasma membrane marker enzyme (K⁺, Mg²⁺-ATPase) was 14- to 17-times higher in the upper (PEG-rich) than in the lower (Dextran-rich) phase, and the reverse was true for marker enzymes (cytochrome c oxidase, EC 1.9.3.1, and antimycin A-resistant NADPH cytochrome c reductase) of intracellular membranes. The ATPase was highly stimulated by the addition of detergent (Triton X-100), so that the isolated plasmalemma vesicles appear tightly sealed and in a right-side-out orientation. Further characterization of the ATPase activities showed a pH optimum at 6.0 in the presence of Mg²⁺. This optimum was shifted to pH 5.8 after addition of K⁺. K⁺ stimulated the ATPase activity below pH 6 and inhibited above pH 6. The ATPase activity was specific for ATP and sensitive to N,N-dicyclohexylcarbodiimide and sodium vanadate, with K⁺ enhancing the vanadate inhibition. The enzyme was insensitive to sodium molybdate, NO⁻₃, azide and oligomycin. No Ca²⁺-ATPase was detected, and even as little as 0.05 m M Ca²⁺ inhibited the Mg²⁺-ATPase activity.     

Sensitivity to drying in vitro of enzymes in mitochondrial subfractions from Vicia faba seed

In order to investigate the persistence of membrane and matrix functions following desiccation, enzymic activities were studied in Vicia faba L. seed mitochondrial subfractions subjected to drying and rehydration in vitro. Mitochondria were prepared after 0, 12 and 24 h of seed imbibition. These were fractionated into inner membranes ("submitochondrial particles"), outer membranes (12 and 24 h only) and the soluble matrix. Enzyme activities associated with the inner membrane and matrix were found to increase several-fold during the first 12 h of imbibition. The two matrix enzymes examined, malate dehydrogenase and glutamate dehydrogenase, were insensitive to in vitro drying at all stages of imbibition. The membrane-bound activities from 12 h and 24 h imbibed material, antimycin A-sensitive NADH: cytochrome c oxidoreductase and (F_o-F₁)-ATPase of the inner membrane and antimycin A-insensitive NADH: cytochrome c oxidoreductase of the outer membrane, were moderately sensitive to dehydration. The F₁-ATPase solubilized from the inner membrane (F_o-F₁) complex was much less sensitive to drying, provided this was done at room temperature.
Mitochondria posessing their outer membranes could not be prepared from dry seed. The antimycin A-sensitive NADH: cytochrome c oxidoreductase from inner mitochondrial membranes of unimbibed seed was extremely sensitive to desiccation in vitro, about 75 to 80% of the activity being lost. This loss could be somewhat reduced by addition of glycerol or sucrose before drying.
It is concluded that uncontrolled desiccation results in major damage to some of the membrane-bound enzymic systems in mitochondria, whereas activities in the soluble fraction are remarkably tolerant of desiccation.     

Fractionation of Isolated Rat CNS Myelinated Axons by Sucrose Density Gradient Centrifugation in a Zonal Rotor

Myelinated axons isolated from rat CNS brain stem by flotation in a buffered sucrose-salt medium were shocked by vigorous homogenization in hypotonie buffer and then fractionated on a 20-40% (wt/wt) linear sucrose gradient in a Beckman Ti-14 Zonal Rotor. After centrifu-gation to equilibrium, the gradient was fractionated on the basis of sucrose density into 13 individual fractions. The distributions of molecular markers related to myelin [(myelin basic protein, 2’3′-cyclic nucleotide 3′-phos-phodiesterase (EC 3.1.4.37), myelin-associated glycopro-tein (MAG)]; microsomes [CDP-choline:l,2 diglyceride cholinephosphotransferase (EC 2.7.8.2)]; mitochondria [cytochrome c oxidase (EC 1.9.3.1), monoamine oxidase (amine:oxygen oxidoreductase, deaminating, EC 1.4.3.4)], and axolemma [acetylcholinesterase (acetylcho-line hydrolase, EC 3.1.1.7), 5′-nucleotidase (5′-ribonu-cleotide phosphohydrolase, EC 3.1.3.5), Na⁺,K⁺-adeno-sine triphosphatase (EC 3.6.1.3), [³H]saxitoxin binding] were examined, as well as the protein composition and morphological appearance of the fractions. The myelin-related markers were most enriched in the 20-26% region of the gradient, although the MAG was broadly distributed throughout the entire gradient. The axolemma-related markers were most enriched in the 28-32% region of the gradient, whereas the microsomal and mitochondrial-related markers were enriched in the 35-40% region of the sucrose density gradient. Mixing experiments utilizing ¹²⁵I-labeled membrane preparations derived from cultured oligodendroglial and astroglial cells indicated that the constituents of the shocked myelinated axons were not significantly contaminated with glial membranes. The morphology of the fraction was consistent with the membrane molecular marker distribution: the light end of the gradient contained multilamellar myelin; fractions in the center of the gradient were enriched in un-ilamellar membrane fragments; the densest regions of the gradient were enriched in mitochondria. The myelin specific proteins were the prominent polypeptides in the 20-25% regions of the gradient, whereas polypeptides having a molecular weight of 50,000 or greater predominanted in the denser regions of the gradient. The significance of the distribution of these membrane markers and the utility of this fractionation procedure are discussed.     

Synaptosomal Membrane-Bound Form of Endopeptidase-24.15 Generates Leu-Enkephalin from Dynorphin 1-8, α- and β-Neoendorphin, and Met-Enkephalin from Met-Enkephalin-Arg6-Gly7-Leu

Brain contains a membrane-bound form of endopeptidase-24.15, a metalloendopeptidase predominantly associated with the soluble protein fraction of brain homogenates. Subcellular fractionation of the enzyme in rat brain showed that 20-25% of the total activity is associated with membrane fractions including synaptosomes. Solubilization of the enzyme from synaptosomal membranes required the use of detergents or treatment with trypsin. The specific activity of the enzyme in synaptosomal membranes measured with tertiary-butoxycarbonyl-Phe-Ala-Ala-Phe-p-aminobenzoate as substrate was higher than that of endopeptidase-24.11 ("enkephalinase"), a membrane-bound zinc-metalloendopeptidase believed to function in brain neuropeptide metabolism. Purified synaptosomal membranes converted efficiently dynorphin1-8, alpha- and beta-neoendorphin into leucine enkephalin and methionine-enkephalin-Arg6-Gly7-Leu8 into methionine enkephalin in the presence of captopril, bestatin, and N-[1-(R,S)-carboxy-2-phenylethyl]-Phe-p-aminobenzoate, inhibitors of angiotensin converting enzyme (EC 3.4.15.1), aminopeptidase (EC 3.4.11.2), and membrane-bound metalloendopeptidase (EC 3.4.24.11), respectively. The conversion of enkephalin-containing peptides into enkephalins was virtually completely inhibited by N-[1-(R,S)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate, a specific active-site-directed inhibitor of endopeptidase-24.15, indicating that this enzyme was responsible for the observed interconversions. The data indicate that synaptosomal membranes contain enzymes that can potentially generate and degrade both leucine- and methionine-enkephalin.     

Fractionation of membrane vesicles. II. A method for separation of membrane vesicles bearing different enzymes by free-flow electrophoresis.

Free-flow electrophoresis was used to subfractionate membrane vesicles from calf thymocyte plasma membranes. The fractionation resulted in a separation of vesicle populations bearing four different enzymes: alkaline nitrophenyl-phosphatase (orthophosphoric-monoester phosphohydrolase (alkalin optimum) EC 3.1.3.1), gamma-glutamyltransferase (EC 2.3.2.2), (Mg2+ + Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) and acyl-CoA:lysophosphatidylcholine acyltransferase (acyl-CoA:1-acylglycero-3-phosphocholine-O-acyltransferase, EC 2.3.1.23). The specific content of cholesterol and total phospholipid coincided with the distribution of membrane-bound protein. However, vesicles migrating towards the cathode had a higher molar ratio of cholesterol to phospholipid (0.75) compared to those migrating to the anode (0.55). Sodium dodecyl sulphate-gel electrophoresis of pooled vesicle fractions also demonstrates distinct differences in their protein pattern. Electron-micrographic thin sections show that the vesicle populations have a similar morphology and size distribution. These results are discussed in terms of heterogeneity of the original thymocytes, contamination with intracellular membranes and a heterogeneous structure of the plasma membrane.     

The common occurrence of ATP diphosphohydrolase in mammalian plasma membranes

In the plasma membranes from several mammalian tissues (including normal and tumor tissues), a Mg2+ (or Ca2+)-dependent ATP phosphohydrolase activity is present in much greater amount than the (Na+ + K+)-ATPase. The ouabain-insensitive activity can be attributed to at least two enzymes, an ATPase (EC 3.6.1.3) and an ATP diphosphohydrolase (EC 3.6.1.5). The ATPase hydrolyzes ATP and other nucleoside triphosphates and is not inhibited by azide. The ATP diphosphohydrolase hydrolyzes both ATP and ADP (and other nucleoside tri- and diphosphates) and the hydrolysis of adenine nucleotides is strongly inhibited by 10 mM azide. The ratios of these two enzymes in the various membranes (as determined by the extent of azide inhibition) vary widely. The ATP diphosphohydrolase accounts for most of the Mg2+ (or Ca2+)-dependent ATP hydrolysis activity of the plasma membranes of liver (mouse), kidney (dog), two mouse sarcomas, and a human astrocytoma (xenograft in athymic mice). The ATPase is more dominant in the plasma membranes from mouse brain and human oat cell carcinoma. The widespread presence of the ATP diphosphohydrolase in plasma membrane from various types of tissues is demonstrated for the first time and is of particular interest in view of its relatively high activity in the plasma membranes of two sarcomas. The membrane-bound ATP diphosphohydrolase is characterized with respect to its metal ion activators, substrates, and inhibitors. These results should facilitate the distinction of this enzyme from other ATP hydrolyzing enzymes of plasma membranes in future investigations.     

Possibility of mitochondrial-cytosolic cooperation in gluconeogenesis from serine via hydroxypyruvate

Intracellular localization of D-glycerate dehydrogenase (D-glycerate : NAD⁺ oxidoreductase, EC 1.1.1.29), one of the enzymes of the pathway for gluconeogenesis from serine via hydroxypyruvate, was studied by differential centrifugation. Almost all enzyme activity was found in cytosol. Since the major activities of two other enzymes, serine : pyruvate aminotransferase (EC 2.6.1.51) and glycerate kinase (ATP : D-glycerate 2-phosphotransferase, EC 2.7.1.31), of the pathway via hydroxypyruvate are localized in mitochondrial inner membrane and/or matrix, the possible localization of D-glyceratedehydrogenase in mitochondria was examined. Detailed analysis of mitochondrial fraction prepared by differential centrifugation indicated that rat liver mitochondria do not contain any D-glycerate dehydrogenase activity. Based on these results, a cooperative connection between mitochondria and cytosol in gluconeogenesis from serine via hydroxypyruvate is proposed. Possible mechanisms for transport of intermediates of the pathway via hydroxypyruvate across the mitochondrial membranes are also discussed.     

Different enzymes involved in NADH- and NADPH-dependent respiration in the cyanobacterium Anabaena variabilis

Abstract Filaments of N₂-grown Anabaena variabilis exhibit soluble NADPH- and membrane-bound NADH-oxidizing activities. The NADPH-specific enzyme has been identified as ferredoxin-NADP oxidoreductase (FNR; EC 1.18.1.2) by the thionicotinamide-NADP transhydrogenase test, a ferredoxin-dependent hydrogenase assay, and by diaphorase systems. The FNR is easily removed by washing of French-press-prepared membranes. Concurrently, a loss of NADPH-dependent respiration is apparent, which is not reconstitutable by addition of Anabaena cytochrome c -553. The NADH-oxidizing activity, however, is only slightly affected by the washing procedure, and is completely reconstituted by cytochrome c -553. NADPH-dependent oxygen uptake is strongly inhibited by NADP, whereas inhibition of NADH-dependent oxygen uptake by NAD is less pronounced. The data give evidence that NADH and NADPH oxidations linked to the respiratory chain are mediated by two different enzymes.     

Separation of the primary dehydrogenase from the cytochromes of the nicotinamide adenine dinucleotide (reduced form) oxidase of Bacillus megaterium

A selective extraction procedure was developed for sequentially extracting a fraction containing the primary dehydrogenase and a fraction containing the cytochromes of the nicotinamide adenine dinucleotide (reduced form) (NADH) oxidase of Bacillus megaterium KM membranes. The primary dehydrogenase (NADH-2,6-dichlorophenolindophenol oxidoreductase) activity was extracted from sonically treated membranes with 0.4% sodium deoxycholate for 30 min at 4 C. The insoluble residue was extracted with 0.4% sodium deoxycholate in 1 m KCl for 30 min at 25 C. A combination of the two extracts and dilution in Mg(2+) gave good recovery of the original membrane NADH oxidase activity. The primary dehydrogenase fraction contained 41% of the membrane protein, no cytochromes, flavine adenine dinucleotide as the sole acid-extractable flavine, and most of the membrane ribonucleic acid (RNA). The cytochrome-containing fraction had 16% of the membrane protein, 61% of the membrane cytochrome with the same relative amounts of cytochromes a and b as the original membrane, no acid-extractable flavine, little RNA, and no oxidoreductase activity. The oxidoreductase fraction remained soluble after removal of deoxycholate whereas the cytochrome fraction became insoluble after removal of deoxycholate-KCl, but the precipitated fraction could be redissolved in 0.4% sodium deoxycholate. Treatment of both fractions with ribonuclease to destroy all of the RNA present did not affect the ability of the fractions to recombine into a functional oxidase unit. Treatment of either fraction with phospholipase A prevented restoration of a functional oxidase when the oxidoreductase and cytochrome fractions were treated in solution, but no affect on restoration of oxidase was observed when the phospholipase A treatment was carried out with the soluble oxidoreductase fraction and the insoluble cytochrome fraction.