Purification and Properties of a Thermophilic Bacteriophage Lytic Enzyme

A phage lytic enzyme was isolated from lysates of Bacillus stearothermophilus (NCA 1503-4R). The enzyme was purified 1,998-fold with a 27% recovery of enzyme activity. By use of polyacrylamide gel electrophoresis and sucrose gradient centrifugation the enzyme was judged free from protein contaminants. The lytic enzyme was active over a pH range of 6.0 to 7.0, with a maximum at 6.3, and it was stable between pH 7.0 and 8.0 and at 5.0 and unstable between pH 5.5 and 6.5. The temperature coefficient (Q(10)) was 2.27 between 35 and 45 C, 2.01 between 45 and 55 C, and 2.00 between 50 and 60 C. Lytic enzyme in 0.1 m sodium phosphate was not inactivated after a 1-hr exposure to temperatures below 65.5 C, whereas a 1% inactivation was observed at 70.6 C. A 2-hr exposure at 60.1, 65.5, and 70.6 C resulted in an inactivation of 1.2, 9.6, and 12.0%, respectively. A sodium phosphate concentration of at least 0.1 m was necessary for the prolonged exposure of lytic enzyme at 55 C (pH 6.3), whereas 0.005 m was required for maximal lytic activity. Lytic activity was stimulated 169, 165, and 160% by 10(-4)m Mg(++), Ca(++), and Mn(++), respectively. Lytic activity was inhibited 75% by 10(-4)m ethylenediaminetetraacetic acid (EDTA). The EDTA inhibition could be reversed by the addition of excess Mg(++), Ca(++), or Mn(++). Lytic activity was not affected by NaCl, KCl, or NH(4)Cl. Lytic activity was inhibited 100, 91, 25, 61, and 56% by 10(-4)m Hg(++), Cu(++), Zn(++), p-chloromercuribenzoate, and p-hydroxymercuribenzoate, respectively. Cysteine or 2-mercaptoethanol did not stimulate lytic activity, nor were these sulfhydryl compounds required for maintenance of enzyme activity during handling or storage. Cell walls were rapidly solubilized when incubated with lytic enzyme. Lytic action was complete after 1.5 min, with a 70% reduction in optical density (OD). Cell walls without lytic enzyme showed no reduction in OD during this period. The solubilization of N-terminal amino groups paralleled the reduction in OD and reached a level of 0.3 mumole/mg of cell wall after 4 min of incubation. Cell walls with and without lytic enzyme treatment showed a 3- and a 1.3-fold increase, respectively, in N-terminal amino groups after 3 hr of incubation. There was no release of reducing power in either the untreated cell wall suspensions or those treated with lytic enzyme. Electron micrographs of treated and untreated cell walls showed that the enzyme partially degrades the cell wall with the release of small wall fragments.     

Regulatory mechanisms of cellular respiration; the role of soluble sulfhydryl groups as shown by the effect of sulfhydryl reagents on the respiration of sea urchin sperm

Oxidizing agents of sulfhydryl groups such as iodosobenzoate, alkylating agents such as iodoacetamide, and mercaptide-forming agents such as cadmium chloride, mercuric chloride, p-chloromercuribenzoate, sodium arsenite, and p-carboxyphenylarsine oxide, added in small concentrations to a suspension of sea urchin sperm produced an increase in respiration. When the concentration was increased there was an inhibition. These effects are explained by postulating the presence in the cells of two kinds of sulfhydryl groups: soluble sulfhydryl groups, which regulate cellular respiration, and fixed sulfhydryl groups, present in the protein moiety of enzymes. Small concentrations of sulfhydryl reagents combine only with the first, thus producing an increase in respiration; when the concentration is increased, the fixed sulfhydryl groups are also attacked and inhibition of respiration is the consequence. Other inhibitors of cell respiration, such as cyanide and urethanes, which do not combine with -SH groups, did not stimulate respiration in small concentration.     

Studies on Cellulose Synthesis by a Cell-free Oat Coleoptile Enzyme System: Inactivation by Airborne Oxidants

Particulate cell wall polysaccharide synthetase from oat coleoptiles could use either guanosine diphosphate glucose or uridine diphosphate glucose; the latter was a much more effective glucose donor. The neutral polymer derived from uridine diphosphate glucose utilization yielded, after cellulase digestion, mostly cellobiose and to a lesser extent a substance tentatively identified as a mixed-linkage β1,4 = β1,3-trisaccharide; only cellobiose was found after guanosine diphosphate glucose utilization. The uridine diphosphate glucose utilizing system was inactivated by peroxyacetyl nitrate treatment of intact tissue and to a lesser extent by ozone treatment suggesting that this system is a possible site of interference with cellulose and non-cellulosic glucan biosynthesis in vivo. Direct treatment of the enzyme in vitro by peroxyacetyl nitrate, iodoacetamide or p-chloromercuribenzoate also inactivated the enzyme, indicating that the mechanism of inactivation possibly involves reaction with sulfhydryl groups.     

Inhibition of the NADP-linked glutamate dehydrogenase from Trypanosoma cruzi by sulfhydryl reagents.

1. The NADP-linked glutamate dehydrogenase purified from epimastigotes of Trypanosoma cruzi was strongly, but not completely, inhibited by sulfhydryl reagents, in the presence of Tris-HCl or phosphate buffers. 2. The enzyme modified by preincubation with o-iodosobenzoate had a kinetic behaviour different from that shown by the enzyme modified with other inhibitors, such as N-ethylmaleimide or p-chloromercuribenzoate. 3. The inhibition by o-iodosobenzoate was additive with the inhibition by the other reagents tested. 4. It is suggested that two or more different sulfhydryl groups, placed probably near the active site, are involved in these effects.     

Competitive inhibition of hexokinase isoenzymes by mercurials.

A difference in the mode of inhibition of hexokinase [EC 2.7.1.1] isoenzymes by p-chloromercuribenzenesulfonate was confirmed with respect to glucose between two Type I isoenzyme preparations purified from the kidney and spleen of rat. Essentially the same difference was observed when galactose was used as the substrate in place of glucose, as the kidney Type I isoenzyme was inhibited in a competitive manner while the spleen counterpart was inhibited in a non-competitive manner by sulfhydryl inhibitor. Both the Type I isoenzymes, however, were competitively inhibited by other mercurial sulfhydryl inhibitors, methyl and butyl mercuric chlorides. On the other hand, the Type II hexokinase isoenzymes purified from the muscle, heart, and spleen were all inhibited competitively by p-chloromercuribenzenesulfonate with respect to glucose. The mechanism of competitive inhibition of the hexokinase isoenzymes by sulfhydryl inhibitors was discussed in view of the difference in the mode of action of the mercurials with different isoenzymes.     

Beta-Glucosidases from Cicer arietinum L. Purification and Properties of isoflavone-7-O-glucoside-specific beta-glucosidases.

Beta-Glucosidases specific for isoflavone 7-O-glucosides have been isolated from garbanzo plants, Cicer arietinum L. These aryl-beta-glucohydrolases occur in the different organs of the plant as multiple molecular forms. The major isoenzymes of the roots, the leaves and the hypocotyl were purified to electrophoretic homogeneity. When subjected to isoelectric focussing in polyac rylamide gels the electrophoretically homogeneous glucohydrolases were found to consist of one or two major and several minor enzymically active molecular species. In roots the beta-glucohydrolase isoenzymes constitute a considerable portion of the extractable protein, so that purification to an electrophoretically homogeneous form is easily attainable. All beta-glucosidases analyzed possess molecular weights in the range of 125 000 (ultracentrifugation) to 135 000 (Sephadex G-200) and contain two subunits of molecular weight near 68 000. The pH optimum for enzymic activity is 7--7.5 with a second optimum of 4.5--5. The isoelectric points of the various species range between pH 5.9 AND 7.1. Staining for glycoprotein was positive. Kinetic analysis demonstrated a pronounced specificity of the enzymes for aromatic substrates with glucose as the sugar moiety. alpha-Glucosides as well as disaccharides were not hydrolyzed at all. Isoflavone 7-O-glucosides are the most favoured substrates with a Km of 2 x 10(-5) M, while the Km with aromatic glucosides (i.e. salicin, 4-nitrophenyl glucoside) are 100 times larger. In addition the beta-glucosidases show a pronounced specificity for glucose in the 7-position of the flavonoid nucleus. Using isoflavone aglycones as substrates glucose transferase activity was also demonstrable. The beta-glucohydrolase activity is strongly inhibited by Hg2plus. This inhibition is partially reversible and preferentially influences the Km values of the enzymes compared to V. Agplus, glucono-1,5-lactone, ethyleneglycol monomethyl ether and glycerol are only weakly inhibitory, while glucose, p-chloromercuribenzoate and Cu2plus are without effect.     

The activation of glucose dehydrogenase by p-chloromercuribenzoate

Summary P-Chloromercuribenzoate alters various reactions of rat liver glucose (hexose phosphate) dehydrogenase differently. The reagent has little effect on the glucose: NAD or the glucose: NADP oxidoreductases, doubles the rates of oxidations of galactose-6-phosphate and glucose-6-phosphate by NADP and greatly stimulates the oxidations of glucose-6-phosphate and galactose-6-phosphate by NAD. The reagent appears to react with a sulfhydryl group of the enzyme since activation is reversed and prevented by mercaptoethanol. The direct reaction of the reagent with the enzyme is indicated by its lower thermal stability in the presence of the p-chloromercuribenzoate. The size of the enzyme appears to be the same when determined by sucrose gradient centrifugation in the presence or absence of p-chloromercuribenzoate. In microsomes, the oxidation of NADH or NADPH hampers measurements of glucose dehydrogenase. Since p-chloromercuribenzoate inhibits microsomal oxidation of reduced nicontinamide nucleotides, it is possible to assay for glucose dehydrogenase accurately in the presence of the mercurial in microsomes and microsomal extracts and thus measure the effectiveness of a detergent in extracting the enzyme from microsomes.Abbreviation pcMB p-chloromercuribenzoic acid     

Isolation of SAU 96 I restrictase, its physical and catalytic properties

Restrictase Sau 96 I was isolated from Staphylococcus aureus PS 96 and purified by chromatography on DEAE-cellulose, phosphocellulose and hydroxylapatite. The preparation was studied by gel filtration on Toyopearl HW-55 and polyacrylamide gel electrophoresis under denaturing conditions. The active form of the enzyme is a dimer with a molecular weight of 54,000 +/- 5000 composed of two identical subunits. Catalytic properties of the restrictase were determine; the pH optimum is 8.5-9.0, the optimal concentration of NaCl and Mg2+ is 15-100 mM and 10 mM, respectively. Mn2+ ions at a concentration of 2 mM can replace Mg2+, while Zn2+, Ca2+, Cu2+ ions cannot replace Mg2+. The optimal temperature is 30-43 degrees. Ethanol and glycerol at concentrations more than 10% inhibit the enzyme without changing its specificity; p-chloromercuribenzoate inhibits the enzyme at a concentration of 0.05 mM.     

Mercuric and cadmium ions stimulate phosphorylation of band 4.2 protein on human erythrocyte membranes

In this study, we found that Hg2+ and Cd2+ enhanced the phosphorylation of human erythrocyte membranous proteins, especially band 4.2 protein, which was hardly phosphorylated in the absence of the metal ions. p-Chloromercuribenzenesulfonate and p-chloromercuribenzoate had effects similar to those of Hg2+ and Cd2+ on band 4.2 protein phosphorylation, while other metal ions and sulfhydryl agents, such as N-ethylmaleimide, 5,5'-dithiobis-(2-nitrobenzoic acid), or iodoacetate, did not. The Hg2+-stimulated phosphorylation of band 4.2 protein required a millimolar concentration of Mg2+, and it was inhibited by Ca2+ dose-dependently. Phosphoserine was identified from a hydrolysate of the phosphorylated band 4.2 protein by high-voltage electrophoresis. A specific protein inhibitor against cAMP-dependent protein kinase decreased the Hg2+-stimulated phosphorylation of band 4.2 protein. This protein had more binding sites for 203Hg2+ than any other membrane proteins. A spectrin complex from the Hg2+-treated membranes contained the band 4.2 protein, which was not detected in the complex from untreated membranes. Furthermore, protein kinase, which could phosphorylate the band 4.2 protein, was also contained in the cytoskeletal fraction from the Hg2+-treated membranes. These results suggest that Hg2+ may bind certain sulfhydryl groups of band 4.2 and other proteins to make band 4.2 protein susceptible to the endogenous cAMP-dependent protein kinase.     

Sulfhydryl groups of an extramitochondrial acetyl-CoA hydrolase from rat liver

An extramitochondrial acetyl-CoA hydrolase (EC 3.1.2.1) purified from rat liver was inactivated by heavy metal cations (Hg2+, Cu2+, Cd2+ and Zn2+), which are known to be highly reactive with sulfhydryl groups. Their order of potency for enzyme inactivation was Hg2+ greater than Cu2+ greater than Cd2+ greater than Zn2+. This enzyme was also inactivated by various sulfhydryl-blocking reagents such as p-hydroxymercuribenzoate (PHMB), N-ethylmaleimide (NEM), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and iodoacetate (IAA). DL-Dithiothreitol (DTT) reversed the inactivation of this enzyme by DTNB markedly, and that by PHMB slightly, but did not reverse the inactivations by NEM, DTNB and IAA. Benzoyl-CoA (a substrate-like competitive inhibitor) and ATP (an activator) greatly protected acetyl-CoA hydrolase from inactivation by PHMB, NEM, DTNB and IAA. These results suggest that the essential sulfhydryl groups are on or near the substrate binding site and nucleotide binding site. The enzyme contained about four sulfhydryl groups per mol of monomer, as estimated with DTNB. When the enzyme was denatured by 4 M guanidine-HCl, about seven sulfhydryl groups per mol of monomer reacted with DTNB. Two of the four sulfhydryl groups of the subunit of the native enzyme reacted with DTNB first without any significant inactivation of the enzyme, but its subsequent reaction with the other two sulfhydryl groups seemed to be involved in the inactivation process.     

Nature of the specificity of alcohol coupling to L-alanine transport into isolated membrane vesicles of a marine pseudomonad

Ethanol stimulated the uptake of l-alanine into isolated membrane vesicles of a marine pseudomonad at a rate and to an extent comparable with that obtained with reduced nicotinamide adenine dinucleotide (NADH) or the artificial electron donor ascorbate-N, N, N', N'-tetramethyl-p-phenylenediamine (ascorbate-TMPD). Methanol and branched-chain alcohols had little or no capacity to energize transport. No quantitative relationship was found between the ability of a compound to induce oxygen uptake and to energize transport, since with ethanol initial rates of oxygen uptake were approximately 4% of that obtained with NADH or ascorbate-TMPD. Cytochrome analysis revealed that NADH and ethanol reduced cytochromes b and c, whereas ascorbate-TMPD coupled primarily at the level of cytochrome c. Approximately 25% of the cytochromes reduced by dithionite were reducible by ethanol. Ethanol reduction of both cytochromes b and c was prevented by 2-heptyl-4-hydroxyquinoline-N-oxide, p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetate. The ethanol- and NADH-energized transport systems for l-alanine were subject to quantitatively similar inhibition by cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide, 2, 4-dinitrophenol, and the sulfhydryl reagents p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetate. In contrast, for ascorbate-TMPD-driven transport, only cyanide and 2, 4-dinitrophenol remained fully effective as inhibitors, p-chloromercuribenzoate was only half as effective, and the other compounds stimulated transport. Inhibition of ethanol oxidation strikingly paralleled the inhibition of ethanol-driven transport for each of the inhibitors, including 2, 4-dinitrophenol. Marked differences between inhibition of oxygen uptake and inhibition of transport were observed when NADH or ascorbate-TMPD were the electron donors. The data indicate that only a small proportion of the respiratory chain complexes in the membrane vesicles are involved in transport and these are efficiently coupled to ethanol oxidation. The results also suggest that when 2, 4-dinitrophenol inhibits transport it is not acting as an uncoupling agent.     

Glucose transport in Streptococcus agalactiae and its inhibition by lactoperoxidase-thiocyanate-hydrogen peroxide.

Transport of 2-deoxyglucose or glucose in Streptococcus agalactiae was strongly inhibited if the cells were first exposed to a combination of lactoperoxidase-thiocyanate-hydrogen peroxide (LP-complex). The inhibition was completely reversible with dithiothreitol. N-ethylmaleimide and p-chloromercuribenzoate inhibited sugar transport, and the inhibition was also reversible with dithiothreitol. Sodium fluoride also inhibited sugar transport. Glucolysis was completely inhibited, and dithiothreitol completely reversed the inhibition. Phosphoenolpyruvate-dependent phosphotransferase activity in S. agalactiae was not strongly inhibited by the LP-complex. Interference of the entry of glucose into cells of S. agalactiae by the LP-complex could well account for its growth inhibitory properties with this organism. The inhibition of glucose transport by the LP-complex and its reversibility with dithiothreitol suggest the modification of functional sulfhydryl groups in the cell membrane as a cause of transport inhibition.     

Effect of sulfhydryl reagents and protease inhibitors on sodium dodecyl sulfate-heat induced dissociation of Ricinus communis agglutinin

Ricinus communis agglutinin dissociated to lower molecular weight forms when heated in sodium dodecyl sulfate in the absence of reducing agents, while ricin was little affected by such treatment. The data suggest that strong noncovalent bonds hold together two A-B heterodimers in the Ricinus communis agglutinin tetramer. Protease inhibitors such as diisopropylfluorophosphate, phenylmethansefulonyl fluoride, and EDTA, did not prevent the sodium dodecyl sulfate-heat induced dissociation; however, sulfhydryl specific reagents (N-ethylmaleimide, 5,5'-dithiobis (2-nitrobenzoic acid) and p-chloromercuribenzoate) were effective. Titration of the lectins in sodium dodecyl sulfate indicated that ricin contains one sulfhydryl and Ricinus communis agglutinin four sulfhydryl groups, none of which react in the presence of 8 M urea. The sulfhydryl groups that could be titrated in the intact proteins in sodium dodecyl sulfate were on the A chains.     

DEGRADATION OF PYRUVATE BY MICROCOCCUS LACTILYTICUS I. : General Properties of the Formate-Exchange Reaction.

McCormick, N. G. (University of Washington, Seattle), E. J. Ordal, and H. R. Whiteley. Degradation of pyruvate by Micrococcus lactilyticus. I. General properties of the formate-exchange reaction (J. Bacteriol. 83:887-898. 1962.-At an alkaline pH, extracts of Micrococcus lactilyticus(2) catalyze the phosphoroclastic degradation of pyruvate to formate and acetyl phosphate and the rapid exchange of formate into the carboxyl group of pyruvate. At an acid pH, hydrogen, carbon dioxide, and acetyl phosphate are produced, and carbon dioxide is exchanged into the carboxyl group of pyruvate. A concentration of approximately 1 m phosphate is required for the phosphoroclastic reaction and formate exchange; the production of carbon dioxide and hydrogen is greatly inhibited by high concentrations of phosphate. Formate exchange requires a divalent metal ion and is stimulated by reducing agents and an atmosphere of hydrogen. Inhibition by p-chloromercuribenzoate, Zn(++), Cd(++), and arsenite indicates that sulfhydryl groups on the enzyme are involved in the reaction; the inhibition by arsenite and Cd(++) may be relieved by 2,3-dimercaptopropanol, suggesting that vicinal dithiols may be required. Inhibition by hypophosphite may reflect a competition with formate for a site on the enzyme.At an alkaline pH, alpha-ketobutyrate is degraded to propionate and formate, whereas alpha-ketoglutarate is fermented to succinate, propionate, carbon dioxide, hydrogen, and formate. Formate is exchanged into the carboxyl groups of alpha-ketobutyrate and alpha-ketoglutarate under these conditions. Only traces of alpha-ketovalerate and alpha-ketoisovalerate are fermented at an alkaline pH and the exchange of formate into these compounds is very low.The addition of viologen dyes under the conditions used for formate exchange causes a reduction of pyruvate, alpha-ketobutyrate, alpha-ketovalerate, and alpha-ketoisovalerate to the corresponding alpha-hydroxy acids.     

Role of cellular redox state and glutathione in adenylate cyclase activity in rat adipocytes.

Adenylate cyclase in rat adipocyte membranes was inactivated as a result of treatment with sulfhydryl oxidants or with p-chloromercuribenzoate as well as by S-alkylating agents. The inhibition of the basal and isoproterenol- or glucagon-stimulated enzyme activity by the oxidants or the mercurial could be reversed by adding thiols to the isolated membranes. The activity of the enzyme paralleled the cellular glutathione (GSH) content. Lowering of intracellular glutathione by incubating the cells with specific reactants resulted in the inhibition of both basal and hormone-stimulated adenylate cyclase activity in the isolated membranes. Activity could be partly restored by supplying glucose to the incubation medium of intact cells. The fluoride-stimulated adenylate cyclase was also inhibited by the oxidants or the sulfhydryl inhibitors. The results suggest that adenylate cyclase may be partly regulated by oxidation-reduction. Thus, a direct relationship between both basal and hormone-stimulated adenylate cyclase activity and the cellular redox potential, determined by the cellular level of reduced glutathione, may be ascribed to the protection of the catalytic -SH groups of the enzyme from oxidative or peroxidative reactions and maintenance of the redox optimum for the reaction.     

Mitochondrial respiratory chain inhibitors modulate the metal-induced inner mitochondrial membrane permeabilization

To elucidate the molecular mechanisms of the protective action of stigmatellin (an inhibitor of complex III of mitochondrial electron transport chain, mtETC) against the heavy metal-induced cytotoxicity, we tested its effectiveness against mitochondrial membrane permeabilization produced by heavy metal ions Cd2(+), Hg2(+), Cu2(+) and Zn2(+), as well as by Ca2(+) (in the presence of P(i)) or Se (in form of Na?SeO?) using isolated rat liver mitochondria. It was shown that stigmatellin modulated mitochondrial swelling produced by these metals/metalloids in the isotonic sucrose medium in the presence of ascorbate plus tetramethyl-p-phenylenediamine (complex IV substrates added for energization of the mitochondria). It was found that stigmatellin and other mtETC inhibitors enhanced the mitochondrial swelling induced by selenite. However, in the same medium, all the mtETC inhibitors tested as well as cyclosporin A and bongkrekic acid did not significantly affect Cu2(+)-induced swelling. In contrast, the high-amplitude swelling produced by Cd2(+), Hg2(+), Zn2(+), or Ca2(+) plus P(i) was significantly depressed by these inhibitors. Significant differences in the action of these metals/metalloids on the redox status of pyridine nucleotides, transmembrane potential and mitochondrial respiration were also observed. In the light of these results as well as the data from the recent literature, our hypothesis on a possible involvement of the respiratory supercomplex, formed by complex I (P-site) and complex III (S-site) in the mitochondrial permeabilization mediated by the mitochondrial transition pore, is updated.     

Purification and Characterization of Extracellular Proteinases of Aspergillus oryzae

The extracellular proteinases of Aspergillus oryzae EI 212 were separated into two active fractions by (NH4)2SO4 and ethanol fractionation followed by diethylaminoethyl-Sephadex A-50 and hydroxyapatite chromatography. The molecular weight was estimated by gel filtration to be about 70,000 and 35,000 for proteinases I and II, respectively. Optimum pH for casein and hemoglobin hydrolysis was 6.5 at 60 C for proteinase I and 10.0 at 45 C for proteinase II, and for gelatin hydrolysis it was 6.5 at 45 C for both enzymes. The enzymes were stable over the pH range 6 to 8 at 30 C for 60 min. The enzyme activity for both the proteinases was accelerated by Cu2+ and inhibited by Fe2+, Fe3+, Hg2+, and Ag+. Halogenators (e.g., N-chlorosuccinimide) and diisopropyl fluorophosphate inhibited proteinase II. Sulfhydryl reagents such as p-chloromercuribenzoate and iodoacetate inhibited proteinase I. Sulfhydryl compounds accelerated the action of both enzymes.     

Accumulation of sulfate labelled with S35 by rat tissue in vitro

Rat kidney slices incubated in vitro may show, in parallel with other shifts in electrolyte content, a striking capacity for accumulating sulfate ion (sulfate labelled with S(35)). The uptake is reversed or reduced by CN(-), cooling to room temperature, and by interference with adequate oxygenation. Under the conditions of the experiment, the presence in the medium of sodium acetate and glucose as substrates was found to be without measurable effect on the accumulation. The extent of sulfate uptake is related to the ionic composition of the medium in which the tissue is incubated, for the uptake occurs optimally only in the presence of a K(+) level of about 0.04 M, and is decreased as the concentration of Na(+) rises. Likewise, when Ca(++), Mg(++), or choline is present in the medium, sulfate accumulation may be depressed. In addition to renal cortex, kidney medulla and liver showed capacity for sulfate accumulation, whereas no convincing evidence for significant uptake was obtained with strips of aorta, colon, or diaphragm.     

A LIGHT-SCATTERING TECHNIQUE FOR THE STUDY OF THE PERMEABILITY OF RAT BRAIN SYNAPTOSOMES IN VITRO

Abstract— (1) Swelling of synaptosomes was measured spectrophotometrically by recording changes in extinction at 520 nm.
(2) Synaptosomes behaved as osmometers in NaCl solutions. When the tonicity of the medium was changed, synaptosome volume changed in accordance with Boyle and van't Hoff's Law. These changes were reversed on restoring the tonicity of the medium.
(3) The rate at which a solute entered the synaptosome was determined from the rate of swelling in the presence of that solute. Permeability of synaptosomes to non-electrolytes was in the order glucose ≪ glycerol < thiourea = formamide < propylene glycol = dimethylsulphoxide.
(4) Synaptosomes were freely permeable to ammonium and acetate ions and impermeable to Ca²⁺, Mg²⁺, PO₄²⁻, SO₄²⁻ and oxalate ions.