Specificity in the interaction of phospholipids and fatty acids with vesicle reconstituted cytochrome P-450. A spin label study

The association of fatty acids, androstane, phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid with purified and phospholipid-vesicle reconstituted cytochrome $\text{P-450}$ was studied by spin labeling. Spin-labeled fatty acids were found to be motionally restricted by cytochrome $\text{P-450}$ in both phospholipid vesicles and in microsomes to a much greater extent than spin-labeled phospholipids. The equilibrium of spin-labeled fatty acid between the bulk membrane lipid and the protein interface could be shifted towards an increased amount in the bulk phospholipid phase by the addition of oleic acid or lysophosphatidylcholine, but not by sodium cholate. Microsomes from different animals showed a variable extent of motional restriction of fatty acids, independent of pretreatment of the animals with phenobarbital or β-naphthoflavone, of cytochrome $\text{P-450}$ content, of the presence of type I and type II substrates for cytochrome $\text{P-450}$. These differences are attributed to the presence of varying amounts of lipid breakdown products in the microsomal membrane such as lysolipids or fatty acids which compete with the externally added spin-labeled fatty acids, or with spin-labeled androstane for the binding to cytochrome $\text{P-450}$. The negative charge of the fatty acid was found to be involved in its association with the protein. Cytochrome $\text{P-450}$ was shown to interact only with a few spin-labeled phospholipid molecules in such a way that the motional restriction of the spin acyl chains can be detected by electron paramagnetic resonance ($\text{τ}{}_{\mathrm{<mtext>R</mtext>}}\text{> 10}{}^{\mathrm{?8}}\text{s}$). The number of associated lipid molecules per protein probably is too small to form a complete shell around the protein. This lipid-protein interaction could be destroyed by the addition of sodium cholate, in contrast to the fatty acid-protein interaction.     

Glycoprotein characteristics of the sodium channel saxitoxin-binding component from mammalian sarcolemma

The saxitoxin-binding component of the excitable membrane sodium channel exhibits glycoprotein characteristics as evidenced by its specific interaction with various agarose-immobilized lectins. The detergent-solubilized saxitoxin-binding component interacts quantitatively with immobilized wheat germ agglutinin and concanavalin A and fractionally with immobilized Lens culinaris hemagglutinin and Ricinus communis agglutinin. These lectins preferentially bind $\text{N-}\text{acetylglucosamine}$ and sialic acid (wheat germ agglutinin), mannose (concanavalin A and Lens cunilaris and galactose (Ricinus communis). Removal of terminal sialic acid residues by neuraminidase markedly decreases binding to immobilized wheat germ agglutinin but uncovers sites capable of interacting with lectins specific for galactose and $\text{N-}\text{acetylgalactosamine}$. $\text{β-N-}\text{acetylglucosaminidase}$, an exoglycosidase has no effect on the binding of the channel protein to wheat germ agglutinin. Similarly, phospholipase C has no effect on binding of the solubilized toxin binding component to this lectin. Neither wheat germ agglutinin nor concanavalin A free in solution alters the number of toxin binding sites or their affinity for toxin. The sodium channel saxitoxin-binding component appears to be a glycoprotein containing terminal sialic acid residues and internal mannose, galactose, $\text{N-}\text{acetylglucosamine}$, and $\text{N-}\text{acetylgalactosamine}$ residues. The toxin binding site is spatially separated from the binding sites for the lectins studied. The effect of these sugar moieties must be considered when evaluating the biophysical parameters of the sodium channel.     

Inhibition by pyridoxal-5'-phosphate of gamma-aminobutyric acid receptor binding to synaptic membranes of cat cerebellum.

The binding of $\text{[}{}^3\text{H]γ-aminobutyric}$ acid to cat cerebellar membranes is $\text{reversibly}$ inhibited in a competitive manner by pyridoxal-5′-phosphate present during the binding assay. Structural analogues of the inhibitor have no such effect. If, on the other hand, the membranes are preincubated with pyridoxal-5′-phosphate followed by the addition of sodium borohydride, a rapid, $\text{irreversible}$ inhibition of subsequent γ-aminobutyric acid binding is observed. Since pyridoxal-5′-phosphate is known to inactivate certain enzymes by reacting with essential lysine residues, the present results suggest that such a lysine residue may be present within the γ-aminobutyric acid receptor.     

Studies on the characterization of human erythrocyte acetylcholinesterase and its interaction with antibodies

Human erythrocyte membranes were solubilized in 5% Triton X-100 and the acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was isolated by affinity chromatography utilizing a specific inhibitor, $\text{trimethyl}\text{-p-}\text{aminophenyl}$ ammonium chloride, bound to Sepharose 4B. After a repeated chromatography acetylcholinesterase was found to be homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunization of rabbits with acetylcholinesterase elicited the formation of an antiserum which gave single precipitin lines with the enzyme on immunodiffusion and rocket, crossed and immuno-electrophoreses. The purified enzyme had a specific activity of 418 units/mg protein. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value of acetylcholinesterase with acetylthiocholine as substrate was $\text{1.5 × 10}{}^{\mathrm{?4}}\text{M}$. Isoelectric focusing of acetylcholinesterase in the presence of Triton X-100 and within the pH ranges of 3–10 and 3–6 exhibited a single peak of enzyme activity with a $\text{P}\text{I}$ of 4.8. The results of amino acid and carbohydrate analyses showed that acetylcholinesterase is a glycoprotein with a carbohydrate/protein weight ratio of 0.16 and glucose, galactose, mannose, glucosamine, galactosamine and sialic acid as the sugar components. The N-terminal amino acid was blocked. Lipid, phosphorus and fatty acid analyses indicated phosphatidylserine and cholesterol as the major lipid components of acetylcholinesterase. The apparent subunit molecular weight estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol was 160 000 and in its presence, 80 000. The kinetic studies showed a competitive inhibition of acetylcholinesterase by its antibodies. Agglutination of human red cells by monospecific antiserum to acetylcholinesterase confirmed that the antigenic site(s) of the enzyme is localized on the outer surface of the erythrocyte membrane.     

The presence of multiple cytochrome b proteins in succinate-cytochrome c reductase.

Two cytochrome $\text{b}$ proteins were isolated from succinate-cytochrome $\text{c}$ reductase and the cytochrome $\text{b-c}{}_1$ complex. Their molecular weights were determined to be 37,000 and 17,000 daltons by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Spectral properties and amino acid composition of these two proteins are reported in the paper.     

Effect of phenethylbiguanide on sugar and amino acid transport in rabbit ileum

Phenethylbiguanide has been shown to be an inhibitor of sugar and amino acid uptake in both $\text{in vivo}$ and $\text{in vitro}$ conditions. This action could be due to a competition for sodium sites on the sugar and amino acid carrier molecules. The effects of phenethylbiguanide on $\text{in vitro}$ intestinal preparations indicate that this compound has a time-dependent effect, it is most effective when placed on the mucosal surface but is also effective on the serosal surface. Furthermore, competition studies indicate that it is a competitive inhibitor of sugar uptake and a non-competitive inhibitor of amino acid uptake. These results are consistent with the differences in the mechanism of coupled transport between sugars and amino acids, but, do not substantiate the idea that phenethylbiguanide competes for the sodium site on the ternary carrier.     

Inhibition of porphobilinogen synthase by heme and an enol-ether amino acid

The enol-ether amino acid, L-2-amino-4-methoxy-$\text{trans}$-butenoic acid (AMTB) is an inhibitor of porphobilinogen synthase (PBG synthase) when added prior to the addition of the substrate δ-aminolevulinic acid. The inhibition of PBG synthase by several stereoisomers and analogues of AMTB was investigated to determine those structural features of AMTB which may be necessary for inhibition. The D-$\text{trans}$ isomer was also an inhibitor after preincubation, whereas the L-$\text{cis}$ isomer inhibited with or without preincubation. The amino acid analogues, DL-vinylglycine, DL-2-aminobutanoic acid, the reduced form of L-2-amino-4-methoxy-$\text{trans}$-3-butenoic acid, L-2-amino-4-(2-aminoethoxy)-$\text{trans}$-3-butenoic acid and its reduced congener did not inhibit PBG synthase even with preincubation. This structure activity relationship indicates that the $\text{trans}$ double bond and methoxy moiety of L-2-amino-4-methoxy-$\text{trans}$-3-butenoic acid are probably required for inhibition.Heme, when preincubated with PBG synthase, was an inactivator of the enzyme. However, when both L-2-amino-4-methoxy-$\text{trans}$-3-butenoic acid and heme were simulatneously preincubated with PBG synthase, inactivation of the enzyme was greater than with either compound separately. The possibility of multiple catalytic sites was suggested by the use of multiple inhibition kinetics in the presence of heme and L-2-amino-4-methoxy-$\text{trans}$-3-butenoic acid.     

Identification of ortho-octopamine and meta-octopamine in mammalian adrenal and salivary gland.

$\text{Ortho}$- and $\text{meta}$- octopamine have been identified in beef and rat adrenal gland and in rat salivary gland by means of gas chromatography-mass spectrometry. The tritrifluoroacetyl derivatives of $\text{ortho}$-, $\text{meta}$- and $\text{para}$- octopamine were resolved by gas chromatography and shown to produce two characteristic ions at $\text{m/e}$ 315 and $\text{m/e}$ 328. The di-O-trimethylsilyl-N-trifluoroacetyl derivatives of these three isomers were also resolved by gas chromatography and shown to produce a characteristic ion at $\text{m/e}$ 267. Biological samples were homogenized in formic acid:acetone, subjected to ion-exchange chromatography and then derivatized. When the derivatized biological extracts were examined for each characteristic ion, peaks were observed at the exact retention times of the standards. The three isomers are present in adrenal gland in concentrations of ～1 μg g^?1 and in rat salivary gland in concentrations of ～0.1 μg g^?1. This evidence confirms a previous report of the presence of $\text{m}$-octopamine in rat salivary gland measured by a radiochemical enzyme assay and is the first report of the presence of $\text{o}$-octopamine in biological tissue.     

Steric analysis of glycerophospholipids by circular dichroism. Stereospecifity of phospholipase D catalyzed transesterification.

The circular dichroism spectra of natural glycerophospholipids and synthetic 1-$\text{sn}$-phosphatidic acid were recorded. 3-$\text{sn}$-phosphatidic acid derivatives were found to show a positive Cotton effect, while 1-$\text{sn}$-phosphatidic acid revealed a negative Cotton effect. The results are interpreted in terms of the carboxyl sector rule. By this method phospholipase D was shown to produce stereospecifically 3-$\text{sn}$-phosphatidyl-1-$\text{sn}$-glycerol when incubated with egg yolk lecithin and exess of glycerol.     

An electron spin probe study of (Na+ + K+)-ATPase-Containing membranes

The modulating effect of membrane lipids on enzyme function has been described by several investigators. We have used the spin probe $\text{N-}\text{oxyl}\text{-4′,4′-}\text{dimethyloxazolidine}\text{-12-}\text{keto}$ methyl stearate (M 12-NSE) to study this interaction in ox brain membranes enriched with $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. This methyl ester of stearic acid is practically insoluble in aqueous media, and consequently spectra of M 12-NSE-labelled preparations are free of “liquid lines”.At least two types of spectra may be obtained when ox brain microsomes are spin labelled with M 12-NSE, indicating the presence of two distinct binding sites. At one site the spin label is relatively unrestricted and gives rise to an isotropic spectrum. A second spectrum, which is obtained from spin label at another site, is similar to that which is observed after incorporation of M 12-NSE into phospholipid bilayers. This suggests that this latter site is within the core of the microsomal membrane.The two binding sites differ in their affinity for the spin probe. The low affinity site is both more abundant in crude preparations and is more easily removed by detergent treatment; spin labels at this site produce isotropic spectra. The high affinity sites are fewer in number and produce broad spectra. In addition these high affinity sites increase in concentration as the enzyme undergoes purification.The two sites are quite distinct in their sensitivity to ascorbic acid, the low affinity site showing a considerably greater rate of reduction by this agent.This study also demonstrates that the delipidation effects of sodium dodecyl sulfate and sodium deoxycholate on $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase-enriched}$ microsomes from ox brain are not identical.It is suggested that the two spin probe binding sites represent two different lipid domains, one of which is very closely associated with the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme and may reflect a protein-directed phospholipid specificity for this enzyme.     

Enhancement of hexobarbital-induced sleep by lysine and its metabolites

Lysine has been shown to be metabolized in the rat brain to pipecolic acid which is a precursor of piperidine. Lysine and its proposed metabolites in this pathway were studied for the first time for their effect on the sleeping time induced by hexobarbital in the rat. Only $\text{L}$-lysine and $\text{D}$-lysine were found to prolong sleeping time significantly without toxic effect. A 3-day pretreatment with $\text{L}$-lysine produced an even more profound sleep prolongation. In most cases sleep enhancement was accompanied by a significant shortening of the time of sleep onset. Quantification of brain hexobarbital levels in the control and treated rats indicates that prolongation of sleeping time was not produced by inhibition of hexobarbital metabolism. The sleep prolonging effect of lysine, therefore, may be a direct action of lysine, or the metabolite(s) derived $\text{in}$$\text{vivo}$ from lysine, on the central nervous system.     

Studies on closure of the ductus arteriosus. XII. In utero effect of indomethacin and sodium salicylate in rats and rabbits

Administration of prostaglandin synthetase inhibitors to pregnant does and dams in late gestation was followed by $\text{in utero}$ contraction of the fetal ductus arteriosus when studied by the whole-body freezing method. In the rat this contraction was well established within 6 h and persisted up to 36 h following 15 mg/kg indomethacin p.o. No effect was observed in the 18 d rat fetus but fetuses at 20 d and 22 d of gestation responded significantly to indomethacin. Doses of indomethacin approaching clinical usage (2.5 mg/kg) also caused a positive response $\text{in utero}$. The rat was found to be sensitive also to sodium salicylate and in the rabbit both indomethacin and sodium salicylate were effective. Exposure $\text{in utero}$ to prostaglandin synthetase inhibitors with resulting contraction of the ductus may seriously disturb cardiac function in the fetus.     

Perturbation of lipid metabolism in L-M cultured cells by elaidic acid supplementation: formation of fatty alcohols

Supplementation of culture medium with elaidic acid (400 μg/flask) in L-M cells results in the formation of an otherwise undetected lipid component. We have identified this lipid component to be a mixture of free fatty alcohols containing primarily elaidyl alcohol with cetyl, stearoyl, and oleoyl alcohols as minor constituents. Formation of fatty alcohols by fatty acid supplementation seems to be specific with $\text{trans}$ fatty acids (i.e., elaidate, $\text{trans}$ vaccenate, and linolelaidate); addition of stearate and oleate to the L-M cells does not produce fatty alcohols. The fatty alcohols accumulated by the $\text{trans}$ fatty acid supplementation are associated with both the particulate and supernatant fractions of the cells.     

Structure of the "keratosulfate-like" material in liver from a patient with G M1 -gangliosidosis ( -D-galactosidase deficiency)

Large amounts of a glycopeptide containing galactose, $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and threonine in the ratio 4:3:1:1, together with smaller amounts of mannose, fucose, sialic acid, sulfate, serine, and other amino acids were isolated from the liver of a patient with G_M1-gangliosidosis. Treatment with mild alkali and sodium borohydride indicated an $\text{O}$-glycosidic linkage between $\text{N}$-acetylgalactosamine and threonine. All the hexosamine residues were resistant to sodium metaperiodate whereas 2 out of 4 D-galactose residues were destroyed. Further studies indicated that one of the galactose residues was 1→3 linked to $\text{N}$-acetylgalactosamine (as in G_M1) and the other 1→4 linked to $\text{N}$-acetylglucosamine as found in skeletal keratosulfate.     

The dependence of the conformation of a (1→3)-β-D-glucan on chain-length in alkaline solution

(1→3)-β-D-Glucans of various degrees of polymerization were prepared by degradation of a gel-forming D-glucan with formic acid. The degraded D-glucans were separated into a water-soluble fraction (soluble D-glucan) and an insoluble fraction (insoluble D-glucan). Both D-glucans were further fractionated. The optical rotation including determination of the o.r.d. curves of the fractions and of the original gel-forming D-glucans was measured at various sodium hydroxide concentrations (0–5M). The results indicate that (1→3)-β-D-glucans of $\text{DP}$n below ca. 25 (the soluble D-glucan) took a disordered form in both neutral and alkaline solutions, whereas the D-glucans of higher $\text{DP}$n (the insoluble and the original D-glucans) took an ordered structure in dilute alkaline solution (0.1M). The proportion of ordered structure in the insoluble D-glucan increases with $\text{DP}$n to attain a maximum value at a $\text{DP}$n of around 200; this may be the lower limit of $\text{DP}$n to permit gel formation in neutral media. The formation of complexes with Congo Red in alkaline solutions by the soluble and the insoluble D-glucans supports the same conclusions.     

Crotoxin effects on Torpedo californica cholinergic excitable vesicles and the role of its phospholipase A activity

The phospholipolytic neurotoxin from $\text{Crotalus}\text{durissus}\text{terrificus}$, crotoxin, is able to produce a dose- and time-dependent block of carbachol-stimulated ²²Na efflux from pre-loaded $\text{Torpedo}\text{californica}$ excitable vesicles. The blocking activity is dependent on calcium and is abolished by chemical modification with p-bromophenacyl bromide. The isolated basic subunit, crotoxin B, produces an identical block, whereas the isolated acidic subunit, crotoxin A, has no detectable effect. Neither crotoxin nor crotoxin B antagonizes the binding of $\text{[}{}^{125}\text{I]-α-bungarotoxin}$ to purified acetylcholine receptor, although, at high concentrations, they antagonize its binding to acetylcholine receptor-rich membrane fragments. Certain phospholipase A₂ enzymes and the fatty acid products of their digestion can mimic the crotoxin action. It is therefore suggested that, although considered a pre-synaptic neurotoxin, crotoxin can have $\text{in}\text{vitro}$ post-synaptic effects, possibly mediated by its endogeneous phospholipase A₂ activity.     

Identification of the outer membrane protein of E.coli that produces transmembrane channels in reconstituted vesicle membranes

Outer membrane of $\text{Escherichia}$$\text{coli}$ allows a rapid diffusion of saccharides of molecular weights less than 550. This permeability property could be restored in vesicle membranes reconstituted from isolated phospholipids, lipopolysaccharide, and an outer membrane protein. The active protein aggregates were isolated from the insoluble material left after solubilization of cell envelope of $\text{Escherichia}$$\text{coli}$ B with sodium dodecyl sulfate at 35°. Analysis by acrylamide gel electrophoresis, isoelectric focusing and amino terminal amino acid determination revealed that only a single species of protein, with a molecular weight of 36,500 forms the oligoprotein aggregates which produces diffusion channels.     

Distribution of free sulfhydryl and disulfide groups among platelet membrane proteins

Reactive sulfhydryl and disulfide groups were identified in platelet membrane proteins resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet membranes treated with $\text{N-}\text{ethyl}\text{[1?}{}^{14}\text{C}\text{]}\text{maleimide}$, phenyl[²⁰³Hg]mercuric acetate and $\text{p-}\text{chloro}\text{[}{}^{203}\text{Hg}\text{]}\text{mercuribenzoate}$ showed similar patterns of distribution of sulfhydryl groups among the sodium dodecyl sulfate-solubilized membrane proteins. Four major and two minor polypeptides ranging in molecular weight from > 200 000 to 20 000 were found to have reactive SH groups. Reduction of membrane proteins by sulfite coupled with subsequent mercaptide formation of the resultant monothiols led to the identification of four polypeptides with disulfide bonds. Reaction of platelet membranes with ¹⁴C-labeled 5,5′-dithio-bis(2-nitrobenzoic acid) resulted in changes in the distribution profile of the solubilized membrane proteins suggestive of a polymerization process dependent upon 5,5′-dithio-bis(2-nitrobenzoic acid)-induced intermolecular disulfide interchange.     

Alterations in labeling of cell-surface glycoproteins from normal and diabetic rat intestinal microvillous membranes

The effect of chronic streptozotocin-induced diabetes was studied on intestinal microvillous membrane surface carbohydrate groups. After 7 weeks of diabetes, purified microvillous membranes were prepared from rat small intestine and surface galactoproteins identified by labeling with galactose oxidase/sodium boro[³H]hydride. Membrane surface sialic acid residues were labeled using the sodium metaperiodate/sodium boro[³H]hydride technique. Membranes were solubilized in SDS and protein labeling analyzed by acrylamide electrophoresis. Membranes from diabetic rats showed an 81% increase in galactoprotein labeling ($\text{P< 0.02}$) while labeling of sialic acid residues was unchanged. The greatest increase in galactoprotein labeling occurred in protein monomers of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 116 000–200 000, where there was a 155% increase in labeling ($\text{P< 0.005}$). These results indicate that intestinal microvillous membrane protein glycosylation is altered in chronic diabetes. This increase in surface membrane carbohydrates could explain the decreased rates of proteolytic degradation previously described for at least one microvillous protein. An increase in membrane galactose groups has also been noted in hepatocyte and kidney glomerular basement membranes, which suggests the presence of a systematic change in membrane protein glycosylation occurring as a result of the diabetic state.