Specific visualization of tubulin-containing structures in tissue culture cells by immunofluorescence. Cytoplasmic microtubules, vinblastine-induced paracrystals, and mitotic figures.

Antibody against tubulin from the outer doublets of sea urchin sperm flagella reacts with tubulin-containing structures in mammalian cells. Thus cytoplasmic microtubules, vinblastine-induced paracrystals and the full spectrum of mitotic figures can be visualized by immunofluorescence. These results show that the tubulin structure has been highly conserved during evolution.     

Separation of hyaluronate, chondroitin sulfate, and heparin by adsorption-desorption technique

An interesting method for separation of the three important mucopolysaccharides, hyaluronate, chondroitin sulfate, and heparin by adsorption to and elution from three inorganic salts Ca₃(PO₄)₂, BaSO₄, and Al₂O₃ has been described in details. Alumina has to be washed with dilute HCl before it can adsorb the mucopolysaccharides, and on treating with alkalies the mucopolysaccharides can be desorbed from it. Calcium phosphate and barium sulfate can adsorb the mucopolysaccharides without any pretreatment. The specific eluents for each of the polysaccharides depend on the nature of the adsorbants also. The recoveries of the mucopolysaccharides are quite satisfactory.     

Morphogenesis of bacteriophage lambda deletion mutants. I. Abnormal head-related structures produced in normal Escherichia coli.

Late in the morphogenesis of bacteriophage lambda, DNA condenses into the nascent head and is cut from a concatemeric replicative intermediate by a nucleolytic function, Ter, acting at specific sites, called cos. As a result of this process, heads of lambda deletion mutants contain less DNA than those of the wild-type phage. It has been reported that phage with very large deletions (22% of the genome or more) grow poorly but that normal growth can be restored by the non-specific addition of DNA to the genome. This finding implies that DNA content may exert a physical effect on some stage of head assembly.We have investigated the effects of two long deletions, b221 and tdel33, on head assembly. Bacteria infected with the mutants were lysed with non-ionic detergent under conditions favoring stabilization of labile structures containing condensed DNA. It has proved possible to isolate two aberrant head-related structures produced by the deletion mutants. One of these (“overfilled heads”) contains DNA which is longer than the deletion mutant genome and is about the same size as that found in wild-type heads. These structures appear to be unable to attach tails. The second type of structure (“incompletely filled heads”) contains a short piece of DNA, 40% of the length of the mutant genome. The incompletely filled heads are found both with and without attached tails. Both of these abnormal structures are initially attached to the replicating DNA but are released by treatment with DNAase. The nature of these abnormal structures indicates that very small genomes affect a late stage of head morphogenesis, after the DNA is complexed with a capsid of normal size. The results presented suggest that underfilling of the capsid interferes with the ability of the Ter function to properly cleave cos.     

Topography of cyclodextrin inclusion complexes : Part II. The iodine-cyclohexa-amylose tetrahydrate complex; its molecular geometry and cage-type crystal structure

Iodine-cyclohexa-amylose tetrahydrate [(C₆H₁₀O₅)₆ ·I₂·d4H₂O] crystallizes in the orthorhombic space-group P2₁2₁2₁, a  14.240 Å, b  36.014 Å, c  9.558 Å. The structure was solved by heavy-atom techniques and refined by least-squares methods to a conventional discrepancy index R  0.148 for the 2872 observed data. The six d-glucose residues are in the C1 chair conformation; the conformational angles vary in magnitude from 45 to 66°, the angles O(5)-C(5)-C(6)-O(6) are close to · 70°, and the six O(4) atoms are almost coplanar (r.m. s. displacement 0.13 Å). Only four of the six O(2) ?O(3) intramolecular hydrogen bonds have formed, which renders the molecule less symmetrical and more conical-shaped than in the previously determined α-cyclodextrin-potassium acetate complex. The iodine molecule is coaxial with the cyclohexa-amylose molecule. The I-I distance is a conventional 2.677 Å. Close interactions between the iodine atoms and the host molecule comprise carbon atoms C(5) and C(6) and oxygen atoms O(4), with interatomic distances all equal to or greater than van der Waals contacts. Intermolecular, almost-linear, short contacts O ? I-I?O with I?O distances of 3.22 and 3.07 Å indicate attractive interaction.The molecules are arranged in herring-bone “cage-type” fashion, with the four water molecules as space-filling mediators; the structure is held together by an intricate network of hydrogen bonds.     

Anaerobic dehalogenation of halothane by reconstituted liver microsomal cytochrome P-450 enzyme system

Cytochrome P-450 from liver microsomes of phenobarbital-treated rabbits catalyzed anaerobic dehalogenation of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) when combined with NADPH and NADPH-cytochrome P-450 reductase. Cytochromes P-450B₁ and P-448 from liver microsomes of untreated rabbits were less active. Triton X-100 accelerated the reaction. Unlike anaerobic dehalogenation of halothane in microsomes, the major product was 2-chloro-1,1,1-trifluoroethane and 2-chloro-1,1-difluoroethylene was negligible. These products were not detected under aerobic conditions, and dehalogenation activity was inhibited by carbon monoxide, phenyl isocyanide and metyrapone.     

Morphogenesis of bacteriophage lambda deletion mutants. II. Escherichia coli mutants which prevent maturation of short genomes.

We have isolated mutants of Escherichia coli which severely reduce the growth of bacteriophage lambda carrying the b221 deletion. Some of the bacterial strains also cause a moderate reduction in the growth of wild-type phage. In the mutant hosts tested, the growth of λb221 is restored by chromosomal alterations producing a non-specific increase in genome length. Thus the defect in growth can be attributed to the physical size of the genome, rather than a genetic effect of the b221 deletion. Our experiments show that the failure to grow results from a block to head morphogenesis and that growth can be restored by mutations in at least two phage head genes. In the accompanying paper we have shown that even in the normal bacterium, the process of packing and cutting the λb221 genome is perturbed as a result of its small size. The block to morphogenesis in the bacterial mutant we have studied most extensively appears to result from an enhancement of the same effect. The experiments described support the hypothesis that there is host participation in the cutting of encapsulated lambda DNA, although it is not yet clear if this involves the direct participation of a host gene product.     

Growth-dependent regulation in production and utilization of acetylated ribosomal protein L7.

The relative levels of protein L12 and its α-N-acetylated form L7 in ribosomes of Escherichia coli have previously been shown to markedly vary during the growth cycle. The present labeling study shows preferential utilization of L12 in early logarithmic phase and of L7 in late logarithmic phase. Both forms are, however, simultaneously used throughout the growth cycle. After assembly into ribosomes, L7 and L12 are conserved without net interconversion. It is therefore concluded that the variation in L12 to L7 ratio takes place through changes in the relative flow of L7 and L12 species into ribosome assembly rather than by modification in pre-existing ribosomes. During this study, we have also measured the surprisingly large difference in the binding of Coomassie Blue to these proteins.     

Spectral properties of a novel cytochrome P-450 of a Saccharomyces cerevisiae mutant SG1. A cytochrome P-450 species having a nitrogenous ligand trans to thiolate

An altered cytochrome P-450 (SG1 P-450) was partially purified from Saccharomyces cerevisiae mutant SG1 which is defective in lanosterol 14 alpha-demethylation. Oxidized SG1 P-450 showed a Soret peak at 422 nm and the alpha peak was lower than the beta peak. This spectrum was considerably different from those of known low-spin P-450s, indicating a unique ligand structure of SG1 P-450. The absorption spectrum of ferric SG1 P-450 was superimposable on that of the imidazole complex of ferric P-450, suggesting the presence of a nitrogenous ligand such as histidine of the apoprotein at the 6th coordination position. SG1 P-450 was immunochemically indistinguishable from cytochrome P-450 of S. cerevisiae catalyzing lanosterol 14 alpha-demethylation (P-45014DM) but had no lanosterol 14 alpha-demethylase activity.     

Correlation of surface antigens and cell type in cloned cell lines from the rat central nervous system

The electrical properties of the clonal muscle cell line L6 can be revealed by the measurement of ion fluxes. Under many circumstances, this technique provides a useful alternative to electro-physiology. In myoblasts, sodium uptake through voltage-dependent ionophores can be stimulated by veratridine and inhibited by tetrodotoxin. In myotubes which result from fusion of myoblasts, these voltage-dependent sodium channels appear to increase in number, paralleling the development of the action potential. Furthermore, in myotubes (but not myoblasts) carbamylcholine is able to stimulate a sodium influx through ionophores which are inhibitable by curare (dTC) but not tetrodotoxin (TTX). This demonstrates the presence of acetylcholine receptors on the fused cells. The cells also have a manganese-inhibitable calcium channel which appears to be voltage dependent and may be responsible for the calcium-dependent component of the action potential. Depolarizing concentrations of potassium in the medium stimulate calcium uptake both in the presence and absence of sodium. Veratridine and carbamylcholine also stimulate calcium influx, but both require the presence of sodium. This indicates that the depolarization necessary for opening the calcium channel is dependent upon sodium influx in these latter cases. Myoblasts and myotubes appear to have these channels in about equal numbers.     

Formation of microsomal cytochrome P-450 complexes studied by the NMR relaxation of water

Cytochrome P-450 in microsomes from liver of phenobarbital treated and control rats has been studied by light absorption and by magnetic resonance methods (EPR and NMR). The nuclear relaxation rate of water protons was measured for microsomal suspensions in the presence of various reactants of Type I and II. The change of relaxation rates correlates well with the spin state conversion of the heme iron. No competition between eventual inner-sphere water molecules and the reactants seems to occur. The temperature dependence of the low spin to high spin equilibrium was studied by light absorption and was accounted for in the temperature variation of the molar relaxation rates of the two spin states.     

Immunochemical evidence for a role of cytochrome P-450 in liver microsomal ethanol oxidation

Antibodies to cytochrome P-450 isozyme 3a, the ethanol-inducible isozyme in rabbit liver, were used to determine the role of this enzyme in the microsomal oxidation of alcohols and the p-hydroxylation of aniline. P-450 isozymes, 2, 3b, 3c, 4, and 6 did not crossreact with anti-3a IgG as judged by Ouchterlony double diffusion, and radioimmunoassays indicated a crossreactivity of less than 1%. Greater than 90% of the activity of purified form 3a toward aniline, ethanol, n-butanol, and n-pentanol was inhibited by the antibody in the reconstituted system. The catalytic activity of liver microsomes from control or ethanol-treated rabbits was unaffected by the addition of either desferrioxamine (up to 1.0 mM) or EDTA (0.1 mM), suggesting that reactions involving the production of hydroxyl radicals from H2O2 and any contaminating iron in the system did not make a significant contribution to the microsomal activity. The addition of anti-3a IgG to hepatic microsomes from ethanol-treated rabbits inhibited the metabolism of ethanol, n-butanol, n-pentanol, and aniline by about 75, 70, 80, and 60%, respectively, while the inhibition of the activity of microsomes from control animals was only about one-half as great. The rate of microsomal H2O2 formation was inhibited to a lesser extent than the formation of acetaldehyde, thus suggesting that the antibody was acting to prevent the direct oxidation of ethanol by form 3a. Under conditions where purified NADPH-cytochrome P-450 reductase-catalyzed substrate oxidations was minimal, the P-450 isozymes other than 3a had low but significant activity toward the four substrates examined. The residual activity at maximal concentrations of the antibody most likely represents the sum of the activities of P-450 isozymes other than 3a present in the microsomal preparations. The results thus indicate that the enhanced monooxygenase activity of liver microsomes from ethanol-treated animals represents catalysis by P-450 isozyme 3a.     

Differences in the mechanism of NADPH- and cumene hydroperoxide-supported reactions of cytochrome P-450

A study has been carried out on the association of aldolase with the human erythrocyte membrane. It has been shown that the conditions employed during hypotonic hemolysis affect the amount of aldolase that remains bound to the cell membrane. Thus, the in vivo nature of this binding cannot be ascertained by this technique. Therefore, a method has been developed in which aldolase is crosslinked with glutaraldehyde to the inner surface of the membrane in intact red blood cells. Under the specified conditions, over 90% of the intracellular aldolase can be crosslinked to the membrane with less than 10% of the hemoglobin becoming bound. These results suggest that the localization of aldolase in situ is on or near the inner surface of the membrane. The amount of aldolase bound to the membrane following crosslinking can be decreased by preincubating the cells with cytoskeletal agents such as cytochalasin B, colchicine, and vinblastine sulfate. The in vitro binding of aldolase to the purified spectrin-actin and F-actin complexes was studied. Aldolase bound both complexes very tightly (K_D ? 10^?9m) and this binding could be inhibited by cytochalasin B, but not by colchicine. A competition binding study was carried out to determine if the binding of aldolase to F-actin involved specific interactions. Neither bovine serum albumin nor cytochrome c significantly inhibited the binding of aldolase to F-actin when each was present at equimolar concentrations with aldolase. However, glyceraldehyde 3-phosphate dehydrogenase inhibited aldolase binding to F-actin and when present at equimolar concentrations with aldolase completely blocked the association. The association of aldolase and other glycolytic enzymes with the erythrocyte membrane is discussed and it is postulated that aldolase could be localized in vivo on the inner surface of the membrane by attachment to actin or a spectrin-actin complex.     

Proton coupling in the ligand-binding reaction of ferric cytochrome P-450 from Pseudomonas putida

Effects of pH on the ligand-binding reactions of ferric heme in cytochrome P-450 from Pseudomonas putida (camphor 5-monooxygenase, EC 1.14.15.1) were studied by using cyanide, N-methylimidazole, pyridine, and ethylisocyanide as ligands. In all cases, affinity of the ferric heme for the ligand was found to increase as pH of the medium was raised from around 6 to 9. Depending on the ligand, the increase was 10- to 1000-fold and the shapes of their pH-affinity curves were remarkably different. Analyses such pH profiles disclosed the presence of a dissociable group in the enzyme with a pK value of approximately 9.5 and that its ionization greatly enhanced the affinity of the heme for ligands. When a dissociable ligand such as hydrogen cyanide and N-methylimidazole was used, the dissociated form of the ligand had a higher affinity toward the heme than the undissociated form. The shapes of the pH-affinity curves were successfully simulated as overlapping curves of ionization reactions of the ligand and the dissociable group. In addition, size of the ligand molecule was shown to be also important in the binding reaction: relatively large molecules such as pyridine, ethylisocyanide, and N-methylimidazole bound to the enzyme in a competitive manner against d-camphor concentration, whereas the binding of a smaller molecule such as cyanide was inhibited by the substrate in a noncompetitive manner. On the basis of these findings, control mechanisms for the ligand-binding reactions of the cytochrome P-450 from P. putida are discussed.     

Radical mechanism of aminopyrine oxidation by cumene hydroperoxide catalyzed by purified liver microsomal cytochrome P-450

Under identical experimental conditions, purified preparations of rabbit liver microsomal cytochrome P-450 and beef heart metmyoglobin were equally effective at stimulating the oxidation of aminopyrine to a free radical species by cumene hydroperoxide. Mannitol had no effect on radical levels produced with either hemeprotein-hydroperoxide system; however, specific ligands of the two hemeproteins, substrates of cytochrome P-450, and phospholipid affected the two systems quite differently. Only the metmyo-globindependent oxidation of aminopyrine was significantly inhibited by fluoride and cyanide. Metyrapone, a specific ligand of cytochrome P-450, and benzphetamine, which was N-demethylated by cumene hydroperoxide only in the presence of cytochrome P-450, inhibited only the cytochrome P-450-stimulated oxidation of aminopyrine. Moreover, only with the solubilized liver hemeprotein was aminopyrine radical generation markedly stimulated by phospholipid. Similar properties of aminopyrine N-demethylation and radical formation by the cytochrome P-450-cumene hydroperoxide system have strongly implicated the radical as a requisite intermediate in product formation. Micromolar concentrations of metyrapone caused parallel inhibition, by at least 50%, of both radical generation and formaldehyde production. These results support a radical pathway of N-demethylation proposed for other hemeprotein-hydroperoxide systems (B. W. Griffin and P. L. Ting, 1978, Biochemistry, 17, 2206–2211), in which the substrate undergoes two successive one-electron abstractions, followed by hydrolysis of the iminium cation intermediate. Thus, for this class of substrates, the experimental data are consistent with the oxygen atom of the product arising from H₂O and not directly from the hydroperoxide, which has been previously proposed as a general mechanism for cytochrome P-450 peroxidatic activities.     

The enhancement of cytochrome P-450 catalyzed methoxyflurane metabolism by a heat-stable microsomal protein

We report the existence of a microsomal, heat-stable, trypsin-sensitive factor that stimulates the O-demethylation of methoxyflurane (CHCl₂CF₂OCH₃) by partially purified preparations of rabbit hepatic cytochrome P-450. The factor is able to stimulate by five to twelve-fold the methoxyflurane metabolizing activity of cytochrome P-450. In contrast, the metabolism of benzphetamine is not affected by the presence of the factor. The factor is inactivated by extraction with methanol, chloroform, butanol and ethanol. It remains intact after treatment with 6M guanidine hydrochloride and is soluble in trifluoroethanol. Thus, the weight of evidence indicates that this factor is a rather hydrophobic protein.     

Turnover of membrane proteins: kinetics of induction and degradation of seven forms of rat liver microsomal cytochrome P-450, NADPH-cytochrome P-450 reductase, and epoxide hydrolase

The in vivo turnover of several rat liver microsomal proteins was studied using techniques designed to maximize antibody recognition specificity and minimize reutilization of radioactive labels. The kinetics of degradation of seven cytochrome P-450 isozymes, NADPH-cytochrome P-450 reductase, and epoxide hydrolase were determined in untreated rats and rats treated with phenobarbital or beta-naphthoflavone. In the cases where induction of these enzymes occurred with the above chemicals, rates of synthesis of the proteins were also estimated. In general, the degradation rates of the different proteins were rather similar to each other, and the effects of phenobarbital and beta-naphthoflavone on these rates were not very great. However, in the case of cytochromes P-450, a general trend was observed in which the heme moiety was degraded more rapidly than the apoprotein. Changes in the rates of synthesis of the individual proteins appear to contribute more to the altered steady-state levels which are expressed than do the rates of degradation, and profiles of steady-state enzyme concentrations predicted by the kinetic constants approximate those observed in vivo.     

Regiospecific hydroxylation of lauric acid at the (omega-1) position by hepatic and kidney microsomal cytochromes P-450 from rainbow trout

Microsomes from liver or kidney of untreated rainbow trout hydroxylated lauric acid specifically at the (omega-1) position. Turnover numbers for liver (2.72 min-1) and kidney (14.1 min-1) were decreased seven- and twofold, respectively, following treatment with beta-naphthoflavone. Laurate hydroxylation activity from untreated trout hepatic microsomes was sensitive to inhibition by SKF-525A, but was not sensitive to metyrapone and only partially inhibited by alpha-naphthoflavone. The temperature optimum of laurate (omega-1) hydroxylation in trout liver microsomes was 25-30 degrees C. The Km and Vmax for (omega-1)- hydroxylaurate formation was 50 microM and 1.63 nmol min-1 mg-1, respectively, in liver and 20 microM and 3.95 nmol min-1 mg-1, respectively, in kidney from untreated trout microsomes. (omega-1) Hydroxylation of laurate, in both liver and kidney microsomes, was sensitive to an antibody raised against a previously purified cytochrome P-450 isozyme (LM2) of trout liver microsomes, which has been shown to be active towards aflatoxin B1. Antibody to the major isozyme of cytochrome P-450 ( LM4b , active towards benzo(a)pyrene) induced by beta-naphthoflavone did not inhibit (omega-1) hydroxylation of laurate in microsomes from untreated or beta-naphthoflavone-treated trout.     

The influence of phospholipid polar groups on gramicidin channels

The influence of well-defined changes in the polar part of phospholipid molecules on the properties of black lipid membranes was studied using a series of phospholipids with identical hydrocarbon chains, but systematically changed polar groups. The hydrocarbon tails of the lipids under study were composed of 1,2-dipentadecylmethylidene glycerol. The polar parts differed in the degree of $\text{N-}\text{methylation}$ and comprised phosphocholine, $\text{-N,N-}\text{dimethylethanolamine}$, $\text{-N-}\text{methylethanolamine}$ and ethanolamine. Stable black lipid membranes could be formed with the solvents octane, decane, dodecane, tetradecane and hexadecane. The properties of gramicidin-induced single ionic channels changed systematically in membranes from the phosphatidylcholine to the phosphatidylethanolamine analogue, as indicated by an increase in the amplitude A of the unit conductance step and a decrease in the average channel life-time or duration τ. The series of τ-values was opposite to that expected from hydrocarbon thickness (specific capacitance). It is suggested that the surface tension γ is a relevant parameter for the prediction of τ-values.     

Convenient procedure for the isolation of highly enriched,cytochrome P-450-containing microsomal fraction from Candida tropicalis

The suitability of Ca²⁺ ions for the precipitation of the microsomal fraction from the hydrocarbon-grown yeast Candida tropicalis was evaluated. In the final procedure the microsomes were precipitated by the addition of 16 mm CaCl₂. Crude extracts obtained from cells via spheroplast lysis were centrifuged at 12,000g for 15 min and at 25,000g for 15 min prior to precipitation. The cytochrome P-450 content of the fraction was between 0.22 and 0.35 nmol mg^?1 protein. The isolated microsomes exhibited both hexadecane hydroxylation activity and NADPH-cytochrome c reductase activity.