A SUBCELLULAR POOL OF HYPO-OSMOTICALLY RESISTANT PARTICLES CONTAINING THYROTROPIN RELEASING HORMONE, α-MELANOCYTE STIMULATING HORMONE, AND LUTEINIZING HORMONE RELEASING HORMONE IN THE RAT HYPOTHALAMUS

Abstract— A 10% homogenate of male rat hypothalami, prepared in 0.32M-sucrose-10μM-CaCl₂, was diluted either with one volume of 0.32M-sucrose-10μM-CaCl₂ (iso-osmotic) or with 10μM-CaCl₂ (hypo-osmotic). A 900 g supernatant fluid fraction (0.9 K-S) was prepared from the diluted homogenates and fractionated on continuous sucrose density gradients under non-equilibrium and equilibrium conditions. In the iso-osmotic 0.9 K-S thyrotropin releasing hormone (TRH), α-melanocyte stimulating hormone (α-MSH), and luteinizing hormone releasing hormone (LHRH) were each found to be sequestered in two populations of particles which were different in size but similar in density. In the hypo-osmotic 0.9 K-S, TRH, α-MSH, and LHRH were each found to be sequestered in a single population of particles. In their sedimentation properties (as judged by differential, non-equilibrium, and equilibrium density centrifugations), the hypo-osmotically resistant particles and the small particles present in the iso-osmotic 0.9 K-S were identical. However, in their peptide content, the two sets of particles differed from each other. If the total quantity of particle-bound peptides recovered after gradient centrifugation of the iso-osmotic 0.9 K-S is taken as 100%, one finds that the amount of TRH, α-MSH. and LHRH recovered in the small particles is 39%, 50%, and 39%. respectively, whereas the amount of TRH, a-MSH, and LHRH recovered in the hypo-osmotically resistant particles is 42%, 68%, and 67%, respectively. This increase in the quantity of peptides sequestered in the small particles occurred concomitantly with the disappearance of peptides from the large synaptosome-like particles. It is estimated that within the large synaptosome-like particles 7% of the TRH, 35% of the a-MSH, and 45% of the LHRH are associated with hypo-osmotically resistant particles. Ultrastructural analysis of purified hypo-osmotically resistant particles containing TRH. α-MSH. or LHRH revealed a predominance of membrane-bounded packets of electron-dense material.     

Calcium transport in isolated bone cells. I. Bone cell isolation procedures

Differential centrifugation of homogenates of Harding-Passey melanoma demonstrated that aryl sulfatase A and β-glucuronidase sediment with particles (i.e., lysosomes) distinct from those particles bearing tyrosinase (i.e., melanosomes). The sedimentation curves for the lysosomal enzymes and tyrosinase, however, demonstrated that an adequate separation of these particle types could not be obtained by differential centrifugation. Isopycnic density gradient centrifugation was used to obtain the necessary resolution. The results of the density gradient studies demonstrated that lysosomes and melanosomes could be separated by this technique, as judged by enzyme distribution among the fractions recovered from the gradients and from electron microscopic examination of the melanosome fractions. It was further evident that the purified and washed melanosomes contained significant amounts of both acid hydrolase activities. Indeed 24% to 27% of the total acid hydrolase activities recovered from the density gradients were associated with the melanosome fractions. The acid hydrolases associated with the melanosomes could not be solubilized by treatment with 0.1% (v/v) Triton X-100, nor by exposure to hypo-osmotic shock. The melanoma lysosomes, however, did release most of both their hydrolase activities into soluble form after treatment with the same percentage of detergent. The lysosomes were, however, very resistant to rupture by exposure to hypo-osmotic conditions.     

Subcellular localization of alpha-melanocyte-stimulating hormone in the rat hypothalamus.

Homogenates of male rat hypothalami were fractionated by means of differential centrifugation, and α-melanocyte-stimulating hormone (α-MSH) in the various fractions was quantified by radioimmunoassay. Of the total quantity of α-MSH in the homogenate, 36% was recovered in the 11,500 g pellet and 31% sedimented between 11,500 and 105,000 g. α-MSH was not detected in the 105,000 g supernatant fluid. When the 900 g supernatant fluid was fractionated on continuous sucrose density gradients at non-equilibrium conditions, two populations of particles containing α-MSH were observed. When fractionated at equilibrium conditions, the two populations were recovered in a single band. These sedimentation characteristics indicate that the particles that contain α-MSH differ in size but are similar in density. After hypo-osmotic shock, the large particles containing α-MSH were not demonstrable, whereas the small particles appeared to be resistant to such treatment. In their sedimentation, the particles containing α-MSH were indistinguishable from particles containing thyrotropin releasing hormone (TRH) but were separable from those that contained luteinizing hormone releasing hormone (LHRH). It is suggested that the large particles containing α-MSH are synaptosomes.     

THE ISOLATION OF CHOLINERGIC SYNAPTIC VESICLES FROM BOVINE SUPERIOR CERVICAL GANGLION AND ESTIMATION OF THEIR ACETYLCHOLINE CONTENT

Abstract— A method is described for the isolation of relatively pure cholinergic synaptic vesicles by hypo-osmotic shock and density gradient centrifugation of a synaptosome fraction prepared from bovine superior cervical ganglia. Vesicles from this source were found to sediment to a density equivalent to 0·3–0·41 M-sucrose. The vesicle subfraction from the gradient had an acetylcholine (ACh) content of 4.4 nmol/mg of protein and were subject to leakage of ACh. By a 'tagging' technique, the vesicles were counted under the electron microscope and their numbers related to their ACh concentration. After correction for leakage, an ACh content of 1630 molecules/vesicle was estimated.     

Unoccupied binding sites for oestrogen in nuclei of a breast tumour cell line (MCF-7).

1. A method to measure both occupied and unoccupied oestrogen receptors directly in the crude nuclear fraction of the MCF-7 cells was developed. The receptors had high affinity for oestradiol (Kd approx. 0.7 nM) and binding specificity characteristics of oestrogen receptors. 2. A substantial amount of the unoccupied receptors were found in the crude nuclear fraction. 3. Several experiments excluded the possibility that the unoccupied nuclear receptor might be a cytoplasmic contaminant. (a) Multiple extractions with Tris buffer released about 75% of the total receptor content, leaving the rest unextractable in the crude nuclear fraction. (b) Nuclei purified by centrifugation through 1.8M-sucrose and treatment with 0.7% Triton X-100, or by centrifugation through 50% glycerol with 0.1% Triton X-100 contained similar amounts of unoccupied receptors to that found in the crude nuclear fraction. (c) In cells cultured during 5 days after preconfluency a 3-fold increase in the amount of unoccupied cytoplasmic receptors occurred, whereas the amount of unoccupied nuclear receptors did not change significantly and conversely in cells exposed to increasing concentrations of oestradiol the unoccupied cytoplasmic receptor was continuously depleted but no considerable change in the unoccupied nuclear receptor was found.     

Phosphatidylglycerophosphate synthease and phosphatidylserine synthase activites in Clostridium perfringens

Cytidine 5'-diphospho (CDP)-1,2-diacyl-sn-glycerol (CDPdiacylglycerol):sn-glycerol-3-phosphate phosphatidyltransferase (EC 2.7.8.5, phosphatidylglycero-P synthase) and CDPdiacylglycerol:L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthase) activities were identified in the cell envelope fraction of the gram-positive anaerobe Clostridium perfringens. The association of phosphatidylglycero-P synthase and phosphatidylserine synthase with the cell envelope fraction of cell-free extracts was demonstrated by sucrose density gradient centrifugation, by both activities sedimenting with the 100,000 x g pellet and solubilization of both activities from the 100,000 x g pellet with Triton X-100. The pH optimum for both enzyme activities was 8.0 with tris(hydroxy-methyl)aminomethane-hydrochloride buffer. Phosphatidylglycero-P synthase activity was dependent on magnesium ions (100 mM). Phosphatidylserine synthase activity was dependent on manganese (0.1 mM) or magnesium ions (50 mM). Both enzyme activities were dependent on the addition of the nonionic detergent Triton X-100. Maximum phosphatidylglycero-P synthase and phosphatidylserine synthase activities were obtained when the molar ratio of Triton X-100 to CDP-diacylglycerol was 50:1 and 12:1, respectively. The Km for sn-glycero-3-P in the phosphatidylglycero-P synthase reaction was 0.1 mM. The Km for L-serine in the phosphatidylserine synthase reaction was 0.15 mM. Both enzyme activities were 100% stable for at least 20 min at 60 degrees C.     

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.     

Uridine phosphorylase activity of isolated plasma membranes of rat liver.

Plasma membranes were isolated from rat liver homogenates either by differential centrifugation or by fractionation in discontinuous sucrose density gradients. Both membrane preparations contained about 17% of the total uridine phosphorylase (EC 2.4.2.3) activity and 44% of the total 5'-nucleotidase (EC 3.1.3.5). The enrichment factor for uridine phosphorylase in the fractions prepared by differential centrifugation was about 2.8 and by the gradient method, as much as 11.0; the respective enrichment factors for 5'-nucleotidase were 1.8 and 9.5. Uridine phosphorylase activity of isolated plasma membrane fractions was stimulated 2.5-fold by 0.1% Triton X-100. Unlike the cytosol enzyme, uridine phosphorylase of plasma membranes showed little or no deoxyuridine-cleaving activity. Contamination of the membrane fractions by thymidine phosphorylase (EC 2.4.2.4) of the cytosol was negligible. The other subcellular organelles obtained by either procedure and characterized by marker enzyme activities were found not to contain significant uridine phosphorylase activity; the cytosol fractions contained just over 70% of the total uridine phosphorylase activity with an enrichment of only about 2.8-fold. The activity of the cytosol enzyme was not stimulated by Triton X-100.     

Acid Sphingomyelinase of Human Brain: Purification to Homogeneity

Abstract: Acid sphingomyelinase (sphingomyelin phosphodiesterase, EC 3.1.4.12) was purified from human brain by extraction with 0.1% Triton X-100, followed by sequential chromatography on Concanavalin A-Sepharose, octyl-Sepharose, hydroxylapatite, DEAE-cellulose, red A-agarose, Sephadex G-200, and DEAE-cellulose with ampholyte elution. Sphingomyelinase activity was purified more than 20,000-fold from the starting homogenate with a 1% yield. Specific activity of up to 800 μmol/h/mg protein could be achieved. Gel electrophoresis with 6% polyacrylamide containing sodium dodecyl sulfate gave a single protein band with a molecular weight of 70,000, in good agreement with the molecular weight previously estimated from sucrose density gradient centrifugation in 0.1% Triton X-100. Triton X-100 could be readily removed from the enzyme by sucrose density gradient centrifugation. The Triton-free enzyme showed the same K _m and pH optimum. Heat stability of the enzyme was reversibly affected by Triton X-100, in that removal of the detergent made the enzyme more heat labile. The K _m of purified enzyme for sphingomyelin was 36 μ M . It was unaffected by sulfhydryl reagents, but was inhibited by dithiothreitol at high concentrations. The preparation was free of all lysosomal hydrolase activities tested, including galactosylceramidase and α-mannosidase, which tended to copurify in our previous procedure. The enzyme was inactive toward sphingosylphosphorylcholine. It was active with bis[ p -nitrophenyll- and bis[4-methylumbelliferyl]phosphate and the chromogenic and fluorogenic sphingomyelin analogues.     

Effects of subcutaneous injection and intrahypothalamic infusion of releasing hormones upon lordotic response to repetitive coital stimulation.

The effects of luteinizing hormone-releasing hormone (LRH) and thyrotropin-releasing hormone (TRH) upon the lordotic response to repetitive coital stimlation were studied using ovariectomized (OVX) and ovariectomized-adrenalectomized (OVX-ADX) female rats. Both OVX and OVX-ADX rats, pretreated with estrone alone, exhibited a dual behavioral response to repeated coital stimulation. The initial response to short-term stimulation was facilitatory with peak sexual receptivity occurring approximately 120 min following the initial male contact. This initial phase was followed by a depression of sexual receptivity associated with continued coital stimulation. Subcutaneous injection of 500 ng of LRH prior to mating was found to significantly potentiate the initial increases in sexual receptivity and to delay the onset of behavioral depression. The injection of 500 ng of TRH was observed to significantly depress behavioral enhancement due to repetitive coital stimulation.The repetitive coital stimulation model was utilized to localize forebrain areas behaviorally responsive to LRH and TRH. Stainless steel cannulas were implanted into either the medial preoptic area (MPOA), arcuate area (ARC), lateral hypothalamic area (LHA), or cerebral cortex (CC). Cannulated animals, primed with estrone, were tested for sexual receptivity immediately prior to experimental treatment, i.e., the infusion of 0.5 μl of 50 ng of LRH or TRH in 0.9% saline, 0.5 μl of 0.9% saline, or sham infusion. A second mating (postinfusion) test was performed 1.75 hr following infusion. When infused into the MPOA or ARC, LRH significantly enhanced lordotic behavior as compared to values obtained for saline or sham infusions. The infusion of LRH into LHA or CC showed no enhancement beyond the levels observed in control infusions (saline and sham infusions). The infusion of TRH into the MPOA or ARC depressed lordotic enhancement to repeated mating, however, this depression was significant only in ARC. These findings were consistent with previously demonstrated actions of releasing hormones upon neural activity within the MPOA and ARC.     

Phosphatidylinositol-glycan-specific phospholipase D is an amphiphilic glycoprotein that in serum is associated with high-density lipoproteins.

Phosphatidylinositol (PtdIns)-glycan-specific phospholipase D was purified from bovine and human serum by phase separation in Triton X-114 and by chromatography on DEAE-cellulose, octyl-Sepharose, concanavalin-A-Sepharose, and hydroxyapatite. The purification of the two enzymes was approximately 1200-fold with a recovery of 3-5%. Bovine serum contained about 40 micrograms/ml of PtdIns-glycan-specific phospholipase D, about 10 times more than the amount determined in human serum. PtdIns-glycan-specific phospholipase D is also present in mammalian cerebrospinal fluid and in mammalian milk but to a much lesser extent than in serum. Enzyme from bovine and human serum displayed amphiphilic properties as revealed by sucrose density gradient centrifugation and gel filtration in the absence and presence of detergent. On density gradient centrifugation, both enzymes sedimented with an apparent sedimentation coefficient of about 6.0 S in the presence of 0.1% Triton X-100, and formed aggregates up to 14.5 S in the absence of detergent. Upon gel filtration, the bovine and human enzymes migrated with a Stokes' radius of 6.5 nm and 6.6 nm, respectively, in the presence of Triton X-100. In the absence of Triton X-100, both enzymes gave a Stokes' radius of 8.8 nm. Serial centrifugation of serum at increasing NaBr concentrations revealed that the majority of the enzyme is contained in the high-density lipoprotein fraction. PtdIns-glycan-specific phospholipase D from bovine and human serum contained 27 and 28 N-acetylglucosamine residues, respectively. Treatment with N-glycosidase F decreased the apparent molecular mass of the bovine and human enzyme from 115 and 123 kDa to 91 and 87 kDa, respectively. Sequence analysis of peptides derived from PtdIns-glycan-specific phospholipase D of bovine serum by CNBr cleavage gave 100% identity to the sequence published for the bovine liver enzyme while there was 83% similarity and 74% identity to the sequence of peptides obtained from the human serum enzyme.     

Subcellular distribution of dopamine in substantia nigra of the rat brain: effects of gamma-butyrolactone and destruction of noradrenergic afferents suggest formation of particles from dendrites.

Abstract— The subcellular distribution of dopamine (DA) in substantia nigra from individual male rats was studied with a fractionation procedure on microscale. After differential centrifugation the distribution of DA coincided with that of noradrenaline (NA) which can serve as a marker for synaptosomes in this area. The proportion of DA/NA concentrations was about 1–2 in most fractions. Sixty per cent of nigral DA was found in P₂ (17,000 g). When P, was layered on a continuous density gradient, DA and NA peaked at the density of 1.0–1.2 M-sucrose. Since DA-containing particles covered a relatively broad range on this gradient, particles between 0.7 and 1.3 M-sucrose were collected with a discontinuous density gradient. Sixty per cent of DA from P₂, was found in this subfraction. The particles containing DA could have been derived from dendrites or axon collaterals of nigrostriatal neurones or represent precursor DA in noradrenergic (NA) terminals. The role of collaterals was investigated by comparing the effect of γ-butyrolactone (GBL, 750 mg/kg, 1 h) on DA concentrations in subcellular fractions from substantia nigra and caudate-putamen. In caudate-putamen, GBL produced a marked increase of DA in total homogenates and subcellular fractions except P₃, whereas DA concentrations remained unchanged in all fractions from substantia nigra. This speaks against a contribution from DA terminals. The proportion of DA contained as precursor in NA terminals was analysed after destruction of the NA input to substantia nigra by two methods. A single injection of 6-hydroxydopamine into the IV ventricle decreased nigral NA by 5574, DA only by 17%. Unilateral electrolytic lesions in the pontine tegmentum affected NA concentrations in homogenates and fraction P₂ of the ipsilateral substantia nigra to a much greater extent than DA. From the results obtained with the two approaches, it is estimated that precursor DA in particulate fractions does not exceed 10%. Our observations indicate that dendrites of the DA neurones in substantia nigra can form particles which behave like synaptosomes on density gradients centrifugation; they may be termed ‘dendrosomes’. According to the proportion of DA found in the particulate fractions at least 4040% of nigral DA appear to be localised in dendrites.     

Solubilization of thyroid peroxidase by nonionic detergents.

We have examined the ability of nonionic detergents to solubilize thyroid peroxidase from a porcine thyroid particulate fraction, as measured by the release of peroxidase activity into the supernatant fraction after centrifugation at 105,000 X g for 1 hour and the retardation of the supernatant peroxidase of Sepharose 6B. The parameters of peroxidase solubilization by Triton X-100 have been investigated in detail. Under optimum conditions, 60 to 95% of the thryoid peroxidase and about 50% of the total protein is released into the 105,000 X g, 1-hour supernatant. Under the optimum conditions established with Triton X-100, a series of Brij detergents of different chemical structure were equally effective in releasing peroxidase and protein. The protein patterns of the supernatants obtained with these detergents were similar on sodium dodecyl sulfate-polyacrylamide electrophoresis gels, suggesting that the detergents studied release similar membrane proteins. The Triton X-100 and Brij 58 supernatants were chromatographed separately on Sepharose 6B equilibrated with 0.1% Triton X-100 or Brij 58, respectively. In both cases, 75 to 80% of the peroxidase activity was retarded, thereby indicating that the nonionic detergents effect solubilization of the peroxidase rather than dispersal of nonsedimentable membrane fragments. These studies report the first successful solubilization of thyroid peroxidase by nonionic detergents. Together with previous evidence from our laboratory, these experiments indicate that thyroid peroxidase is an integral membrane protein.     

Membrane-bound D-gluconate dehydrogenase from Pseudomonas aeruginosa. Purification and structure of cytochrome-binding form.

A membrane-bound D-gluconate dehydrogenase [EC 1.1.99.3] was solubilized from membranes of Pseudomonas aeruginosa and purified to a homogeneous state with the aid of detergents. The solubilized enzyme was a monomer in the presence of at least 0.1% Triton X-100, having a molecular weight of 138,000 on polyacrylamide gel electrophoresis or 124,000--131,000 on sucrose density gradient centrifugation. In the absence of Triton X-100, the enzyme became dimeric, having a molecular weight of 240,000--260,000 on sucrose density gradient centrifugation. Removal of Triton X-100 caused a decrease in enzyme activity. Enzyme activity was stimulated by addition of phospholipid, particularly cardiolipin, in the presence of Triton X-100. The enzyme had a cytochrome c1, c-554(551), which might be a diheme cytochrome, and it also contained a covalently bound flavin but not ubiquinone. In the presence of sodium dodecyl sulfate, the enzyme was dissociated into three components with molecular weights of 66,000, 50,000, and 22,000. The components of 66,000 and 50,000 daltons corresponded to a flavoprotein and cytochrome c1, respectively, but that of 22,000 dalton remained unclear as to its function.     

Monolayer cultures of dispersed cells from bovine anterior pituitary: responses to synthetic hypophysiotropic peptides.

Cells were dispersed from bovine anterior pituitary glands, by digestion with collagenase, and cultured. After 4 days the cell monolayers were incubated with fresh medium containing synthetic hypophysiotropic peptides for 2, 6, or 20 h, and hormone released into the medium was estimated by radioimmunoassay. After 2 h, thyroid releasing hormone (TRH) stimulated the release of thyroid-stimulating hormone (TSH) up to eightfold, and of prolactin (PRL) and follicle-stimulating hormone (FSH) about twofold at a minimal effective concentration of 1 ng/ml; enhanced growth hormone (GH) release was not apparent until 20 h, and release of luteinizing hormone (LH) and adrenocorticotrophic hormone (ACTH) was unaffected. Luteinizing hormone releasing hormone (LH-RH) enhanced release of LH maximally (three- to fourfold) during a 2 h incubation and was effective at 0.1 ng/ml; FSH release was significantly enhanced by about 50% above control level. Growth hormone release inhibiting hormone (GH-RIH)(somatostatin) showed significant effects only in the 20 h incubation; GH release was inhibited by 50% and release of PRL was slightly, but significantly, enhanced. Pituitary cell monolayers apparently permit maximal expression of releasing activities inherent in the hypothalamic hormones.     

Submicrosomal localization of prolyl hydroxylase from chick embryo limb bone.

A fraction greatly enriched in microsomes was prepared from chick embryo limb bone tissue homogenates by differential centrifugation in a high density solution of Metrizamide. This fraction was used to determine the submicrosomal localization of prolyl hydroxylase. At a low concentration (0.05%) of the non-ionic detergents Triton X-100 and Brij-35, 90 to 93% of prolyl hydroxylase activity was released from microsomes. Concentrations of Triton X-100 greater than 0.1% were required to solubilize the intrinsic membrane enzyme NADH-ferricyanide reductase and to release membrane-bound ribosomes, while Brij-35 did not extensively solubilize membrane components even at concentrations up to 0.4%. In addition, prolyl hydroxylase activity which could subsequently be released from microsomes by Brij-35 was relatively resistant to trypsin proteolysis at concentrations which removed more than 50% of the ribosomes and approximately 40% of the protein from microsomes. These results suggest that 90 to 93% of prolyl hydroxylase activity in connective tissue is located within the cisternae of the endoplasmic reticulum. Gel filtration of prolyl hydroxylase released from microsomes or found in the soluble fraction of limb bone homogenates revealed two peaks of activity corresponding to molecular weights of 230,000 and 450,000 to 500,000. The latter is twice the value reported for purified chick embryo prolyl hydroxylase. A fraction of the total prolyl hydroxylase activity (generally 20 to 35%) in microsome preparations could be measured in the absence of detergent, although the microsomal membrane should be impermeable to the large unhydroxylated collagen chains used as substrate. On the basis of experimental data, it was concluded that detergent-independent activity was most likely due to damaged microsomal membranes and that this damage was sufficient to allow substrate and trypsin to enter the cisternae but not to allow prolyl hydroxylase to be released.     

Multiple molecular forms of purified human erythrocyte acetylcholinesterase.

1. Human erythrocyte acetylcholinesterase was solubilized by Triton X-100 and purified by affinity chromatography to a specific activity of 3800 IU/mg of protein. The yield of the purified enzyme was 25--45%. 2. Gel filtration on Sepharose 4-B in the presence of Triton X-100 revealed one peak of enzyme activity with a Stokes' radius of 8.7 nm. Density gradient centrifugation in 0.1% Triton X-100 showed one peak of enzyme activity with an S4 value of 6.3S. 3. Isoelectric focusing in Triton X-100 resolved the enzyme into five molecular forms with isoelectric points of 4.55, 4.68, 4.81, 4.98 and 5.18. Upon incubation with neuraminidase the enzyme activity in the first four forms was decreased with a concommitant increase in activity in the form with the higher isoelectric point. 4. After removal of excess Triton X-100 on Bio-Gel HTP, polyacrylamide gel electrophoresis showed seven bands of protein and corresponding bands of enzyme activity. Density gradient centrifugation of the detergent-depleted enzyme at high ionic strength revealed five multiple molecular forms with S4 values of 6.3 S, 10.2 S, 12.2 S, 14.2 S and 16.3 S. At low ionic strength, higher aggregates were observed in addition to the other forms. Dodecylsulfate-polyacrylamide gel electrophoresis gave one subunit only with an apparent molecular weight of 80 000. 5. These results suggest that human erythrocyte acetylcholinesterase, solubilized by Triton X-100, exists in various forms differing in net charge but of apparently similar molecular dimensions. After removal of the detergent, forms with different molecular sizes are observed.     

The Fate of L-Tyrosine-UL−14C in Shoot-Forming Tobacco Callus

Tobacco callus fed L-tyrosine-UL^?14C was sampled at 3-day intervals for 15 days, homogenized and studied with respect to distribution of incorporated radioactivity. The supernatant obtained by centrifuging of the homogenates at 270 g contained the bulk of the radioactivity although significant activity was also detected in the pellet. Sucrose density gradient centrifugation of the supernatant showed over 90% of the recovered label to be associated with a fraction designated as “less dense than mitochondria”, with the remainder being found in the fraction identified as “mitochondria”. During tissue culture, virtually all of the radioactivity in the fraction “less dense than mitochondria” was recovered in the supernatant obtained by centrifugation at 100,000 g. From 4 to 18% of the labeling in the 100,000 g supernatant fraction was attributable to tyrosine-containing protein, and the rest to free tyrosine and unidentified anionic constituents. The highest proportions of radio-activity in the 270 g pellet were associated with substances extractable with NaCl, pronase, 4.6 N NaOH, and acetolyzing reagent. Low but substantial labeling characterized the extracts obtained with Triton X-100 and 1 N NaOH. The final unextractable residue contained 20% of the 270 g pellet radioactivity.     

Determination of lysosome membrane stability]

Effect of different concentration of non-ionic detergents (Triton X-100, Triton X-305, BRIJ-35 and Triton WR-1339) on total and non-sedimentable activity of 8 rat liver lysosome enzymes (acid phosphatase, acid DNase, acid RNase, arylsulphatases A and B, beta-glucuronidase, beta-galactosidase, beta-glucosidase and beta-acetylglucosaminidase) was studied. Only Triton X-100 at the concentration of 0.1% (and higher) was found to release completely lysosome enzymes. Low concentrations of Triton X-100 (0.025-0.05%) were used to characterize the strength of enzyme binding: the level of releasing acid DNase, beta-galactosidase, beta-glucuronidase and acid phsophatase being considerably higher than that of other lysosome enzymes studied. On the basis of the data obtained a method is worked out, which is suitable for series studies of the stability of lysosome membranes under different physiological and pathological conditions. The essence of the method is the treatment of membrane particles with increasing concentrations of Triton X-100 (0.025; 0.05 AND 0.1%) AND THE SUCCESSIVE ESTIMATION OF NON-Sedimentable activity of marker enzymes. The method detected troubles in the stability of rat liver lysosome membranes under starvation, protein deficiency and aging.     

Assay and subcellular localization of the arylsulphatases in rat brain

—The properties and subcellular localization of type I (nitrophenyl) and type II (nitrocatechol) arylsulphatases were investigated in brain tissue of the rat, and optimal assay conditions were established. Sulphate, phosphate and sulphite ions inhibited the nitrocatechol sulphatases; nitrophenyl sulphatase was inhibited only by sulphite. The presence of latent enzyme activity was demonstrated for the nitrocatechol sulphatases, beta-glucuronidase, and beta-glycerophosphatase in rat and mouse brain homogenates. These hydrolases were highly sensitive to mechanical and osmotic damage; and Triton X-100 was very effective in releasing their latent (bound) activities, a finding suggestive of a lysosomal localization. Activity of nitrophenyl sulphatase was unaffected by osmotic changes or Triton X-100, characteristics suggesting a membranous association for this enzyme. Total activity of nitrophenyl sulphatase was approximately twice as great in canine gray matter as in canine white matter; the converse obtained for beta-glucuronidase activity. Values for total enzymic activity of the nitrocatechol sulphatases in canine white and gray matter were similar. Fractionation of homogenates from rat brain by differential centrifugations and separation of crude mitochondrial fractions by sucrose density gradient centrifugations revealed the following: (1) most of the nitrocatechol sulphatase activity (93 per cent) and all of the nitrophenyl sulphatase activity were sedimentable; (2) crude mitochondrial fractions exhibited the highest relative specific activity (RSA = 1·38) for the nitrocatechol sulphatases, whereas microsomal fractions displayed the highest RSA for nitrophenyl sulphatase (1·89); (3) the lightest fraction (A + B) and the densest fraction (E) from the sucrose density gradient contained most of the activity for both the type I and type II arylsulphatases, whereas the RSA of cytochrome oxidase was greatest in the intermediate density regions (fractions C and D); (4) the highest RSA for beta-glucuronidase and beta-glycerophosphatase occurred in gradient fraction C; (5) appreciable activity of beta-glycerophosphatase was found in a nerve ending fraction (M₃). It is suggested that the hydrolases in heterogeneous tissue like brain might be associated with lysosomal particles of differing enzyme compositions and varying populations, and that the data on distribution lend credence to the concept of bimodal and possible trimodal particle affinity for the hydrolases of brain tissues.