Effect of sodium deoxycholate on 5′-nucleotidase

The 5′-nucleotidase localized in rat liver plasma membranes was purified to a single protein, which contained phospholipid. The molecular weight and the sedimentation constant were about 150 000 and 7 S in the presence of sodium deoxycholate, while the enzyme protein was aggregated when the preparation was dialyzed thoroughly. The purified 5′-nucleotidase exhibited the same properties as the 5′-nuelcotidase in plasma membranes. The 5′-nucleotidase activity was increased by the addition of various bile salts or by the solubilization of membranes with trypsin, papain or phospholipase C. The solubilized and aggregated forms of the enzyme showed different substrate specificity for nucleotides, pH optimum, heat stability and $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. The purified enzyme catalyzed an exchange reaction between AMP and adenosine, which was diminished by the addition of sodium deoxycholate.     

Ligand-dependent tryptic inactivation of the ouabain sensitivity of ADP-ATP exchange catalyzed by canine renal Na+, K+-ATPase.

Phosphate transporter of bovine heart mitochondria was purified by solubilization of submitochondrial particles with octylglucoside and fractionation of the extract with ammonium sulfate. After reconstitution into liposomes the purified protein catalyzed phosphate transport which was sensitive to mersalyl and other SH reagents. Transport measured either as $\text{P}{}_{\mathrm{i}}\text{OH}$ or $\text{P}{}_{\mathrm{i}}\text{P}{}_{\mathrm{i}}$ exchange was proportional to protein concentration and time. The $\text{P}{}_{\mathrm{i}}\text{OH}$ but not the $\text{P}{}_{\mathrm{i}}\text{P}{}_{\mathrm{i}}$ exchange was stimulated several fold by valinomycin plus nigericin in the presence of K⁺. The reconstituted system provides a suitable assay during purification of the mitochondrial phosphate transporter.     

A gel-electrophoretically homogeneous preparation of cytochrome P-450 from liver microsomes of phenobarbital-pretreated rabbits

Cytochrome P-450 was purified from liver microsomes of phenobarbital-pretreated rabbits to a specific content of 16 to 17 nmoles per mg of protein with a yield of about 10 %. The purified cytochrome yielded only a single protein band on sodium dodecylsulfate-urea-polyacrylamide gel electrophoresis, and an apparent molecular weight of about 45,000 was estimated for the protein. The preparation was free of cytochrome $\text{b}{}_5$, NADH-cytochrome $\text{b}{}_5$ reductase, and NADPH-cytochrome $\text{c}$ reductase activities. Aniline hydroxylase and ethylmorphine N-demethylase activities could be reconstituted upon mixing the purified cytochrome with an NADPH-cytochrome $\text{c}$ reductase preparation (purified by a detergent method) and phosphatidyl choline.     

Isolation of bovine cytochrome c1 as a single non-denatured subunit using gel filtration or high pressure liquid chromatography in deoxycholate

The non-denatured cytochrome $\text{c}{}_1$ subunit of bovine ubiquinone-cytochrome $\text{c}$ reductase was isolated using either gel filtration or high pressure liquid chromatography in 1% deoxycholate. The preparation was a single band on polyacrylamide gel electrophoresis in dodecyl sulfate, had a heme content of 31 nmol heme/mg protein, had an absorbance ratio $\text{A}{}_{417}\text{A}{}_{278}\text{= 2.65}$, a visible spectrum with maxima at 553, 530, 523.5, 417, 317, and 277 nm for the reduced protein, and an amino acid analysis identical to that previously reported for the isolated denatured protein. The Stokes' radius of this non-denatured deoxycholate solubilized protein was 34Å, indicating that the protein either is a dimer in deoxycholate, is asymmetric, or binds large amounts of detergent.     

Purification and characterization of a glycoprotein,from normal human liver,which has the same molecular weight and chemical properties as plasma α1-antrypsin

An $\text{α}{}_1\text{-}\text{mantitrypsin-like}$ material has been purified to homogeneity from the soluble fraction of normal human liver by procedures adapted from those employed for plasma $\text{α}{}_1\text{-}\text{antrypsin}$. The liver material, in contrast to a previous report¹ has the same molecular weight as the corresponding normal plasma $\text{α}{}_1\text{-}\text{antrypsin}$. The subunit structure, immunoelectrophoretic and immunological properties of the liver glycoprotein are identical to those of normal plasma $\text{α}{}_1\text{-}\text{antrypsin}$. Amino acid and carbohydrate compositions of the liver material are similar to those of $\text{α}{}_1\text{-}\text{antrypsin}$ obtained from the plasma. The $\text{α}{}_1\text{-}\text{antrypsin-like}$ material has also been isolated and purified from the microsomal fraction of liver It has the same molecular weight and immunological properties as glycoprotein obtained from the cytosol. Although inhibitors of lysosmal proteases were added during the homogenization of the liver, the purified glycoprotein is devoid of trypsin-inhibitory capacity. The loss of inhibitory activity could be due to extensive cellular autolysis before autopsy.     

Purification of a water-soluble Mg2+-ATPase from human erythrocyte membranes

A water-soluble Mg²⁺-ATPase previously reported (White, M.D. and Ralston, G.B. (1976) Biochim. Biophys. Acta 436, 567–576) has been purified from human erythrocyte membranes. The purified enzyme has a molecular weight of 575 000; the apparent minimum molecular weight was 100 000, corresponding to a soluble protein of the component 3 region. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for ATP was 1 mM and apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg²⁺ was 3.6 mM. By means of histochemical activity staining in acrylamide gels it was shown that the purified ATPase preparation could be inhibited by Cd²⁺ and Zn²⁺ salts, $\text{p-}\text{chloromercuribenzoate}$ and $\text{N-}\text{ethylmaleimide}$, known inhibitors of membrane endocytosis.     

Immunochemical study on the participation of cytochrome b5 in drug oxidation reactions of mouse liver microsomes.

Highly purified divalent and monovalent antibodies against cytochrome $\text{b}{}_5$, anti-$\text{b}{}_5$ immunoglobulin G (IG) and anti-$\text{b}{}_5$ Fab', were used in elucidating the role of this cytochrome in the drug-oxidizing enzyme system of mouse liver microsomes. Anti-$\text{b}{}_5$ IG strongly inhibited not only NADH-supported but also NADPH-supported oxidation of 7-ethoxycoumarin and benzo(a)pyrene, but had no inhibitory action on the oxidation of aniline. Anti-$\text{b}{}_5$ Fab' also inhibited NADH-supported and NADPH-supported benzo(a)pyrene hydroxylation. These observations indicate an essential role of cytochrome $\text{b}{}_5$ in the transfer of electrons not only from NADH but also from NADPH to cytochrome P-450 in the microsomal oxidation of some drugs, but not of aniline.     

Alkaline phosphatase. A dominant enzyme of microvillus structure of cephalopod photoreceptors

The rhabdomeres of cephalopod photoreceptors, which are built up mainly of rhodopsin and phospholipid molecules, show a very high alkaline phosphatase activity. The enzyme has been partially characterized in purified rhodopsin vesicle fractions of the rhabdomeres by the following kinetic data: pH optimum 8.7; activation energy 9100 cal·m^?1; $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{= 2.5}$ μmol·min^?1·mg^?1; $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 1.5·10}{}^{\mathrm{?4}}\text{M}$; its activity depends on Mg²⁺. There is good evidence that the alkaline phosphatase is a membrane-bound enzyme with receptor sites presumably located on the inside of the membrane. This enzyme has not been purified but its high activity compared to that of other known alkalin phosphatases (see Table I) indicates that each mirovillus, the structural unit of the rhabdomere, contains 1–20 enzyme molecules. This finding supports the hypothesis that the alkaline phosphatase is involved in the biochemical amplification process of excitation, or adaptation.     

Constraint on the substrate cytochrome P-450 binding reaction in bovine adrenocortical microsomes at physiological temperature

The addition of cholate to the microsomes at 37.5°C resulted in a striking decrease in the apparent substrate dissociation constant ($\text{K′}{}_{\mathrm{<mtext>s</mtext>}}$) and its temperature dependency. The microsomal membranes depleted of 80% of the lipids preserved the temperature dependency of the $\text{K}{}_{\mathrm{<mtext>s</mtext>}}$ and exhibited breaks in the Van't Hoff plot at the characteristic temperature of the lipids phase transition. The results indicate that the cytochrome $\text{P-450}$ is considerably restrained from expressing its maximum substrate binding potential at physiological temperature. In addition, the results indicate that the majority of the lipids apparently do not play a significant role in imposing constraint on the substratecytochrome $\text{P-450}$ binding reaction and in the temperature dependency of the $\text{K}{}_{\mathrm{<mtext>s</mtext>}}$.     

Studies on the mechanism of activation of mitotic histone H1 kinase

A chromatin-associated histone H1 kinase has been detected in synchronized Novikoff hepatoma cells. Enzyme specific activity increased 4 to 6-fold from late G-2 to mid-metaphase, then decayed exponentially ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, 28.5 min) to the interphase level. Extracts of the mitotic kinase retained the ability to decay $\text{in}\text{vitro}$ at 37°C but not at 0°C ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, 24 min), under conditions in which interphase activity was stable. Sedimentation rates in sucrose density gradients of interphase and mitotic enzymes (before and after decay) were identical. Purification did not alter the rate of enzyme decay. However, high ionic strength prevented decay of crude but not purified preparations of mitotic enzyme. The results are discussed in terms of an allosteric mechanism for reversible activation of enzyme activity.     

Purification of bovine liver microsomal NADH-cytochrome b5 reductase using affinity chromatography

Microsomal NADH-cytochrome $\text{b}{}_5$ reductase has been purified from bovine liver by an improved procedure which employs affinity chromatography on ADP-agarose in combination with anion exchange chromatography. The reductase was extracted from a 105,000 × $\text{g}$ microsomal pellet with Triton X-100. The overall purification from isolated microsomes was 98-fold and the yield was 10%. The preparation was nearly homogeneous on SDS-PAGE. This procedure requires less time and effort than previously described procedures. Partially purified cytochrome $\text{b}{}_5$ is also obtained.     

Non-sterol metabolism of mevalonate in vitro: artifacts and realities.

Commercial [5-¹⁴C]mevalonate is shown to contain several radioactive impurities, which give artifactually high amounts of Hyamine bound, volatile acidic radioactivity when incubated with killed or living rat renal cortex slices, as compared with [5-¹⁴C]mevalonate purified either by liquid-liquid partition chromatography or through the enzymically generated $\text{R}\text{-5-phospho-[5-}{}^{14}\text{C]mevalonate}$ by ion-exchange chromatography. The artifactual ${}^{14}\text{CO}{}_2$ results were not diluted by incubation with increasing amounts of unlabelled mevalonate, whereas the ${}^{14}\text{CO}{}_2$ and [${}^{14}\text{C}$]cholesterol produced by rat renal cortex slices incubated with purified [5-¹⁴C]mevalonate were both diluted to the same extent by unlabelled mevalonate. It is concluded that $\text{R}\text{[5-}{}^{14}\text{C]mevalonate}$ is genuinely oxidized to ${}^{14}\text{CO}{}_2\text{in}\text{vitro}$, and that purification of substrate before its use is necessary. Production of ${}^{14}\text{CO}{}_2$ and various [${}^{14}\text{C}$]lipids from purified [5-¹⁴C]mevalonate, as a function of time and substrate concentration, by renal cortex and liver slices, is described.     

Binding of bovine cytochrome b5 to phosphatidycholine liposomes Characterization of the reconstituted lipid-protein vesicles

Cytochrome $\text{b}{}_5$ was extracted and purified from beef liver by a detergent method (cytochrome $\text{d-b}{}_5$). The hydrophilic moiety which carries the heme group (cytochrome $\text{t-b}{}_5$) was prepared by trypsin action upon pure cytochrome $\text{d-b}{}_5$.Single-shelled lecithin liposomes form complexes with cytochromes $\text{d-b}{}_5$ up to a molar ratio of one protein for 35 phospholipids. The lipid-protein complexes were isolated by gel filtration on Sepharose 4B. They are hollow vesicles in which [³H]-glucose can be trapped. Their diameter is greater than that of the initial liposomes.Cytochrome $\text{t-b}{}_5$ does not interact with the vesicles. These results show that the hydrophobic tail is necessary for the binding and that the hydrophilic part of the protein is located on the outer face of the vesicles. This asymmetry is also proved by the action of reducing agents.Experiments with saturated phosphatidylcholines show that the protein interacts with the lipids both below the transition temperature $\text{T}{}_{\mathrm{M}}$. i.e. when the aliphatic chains are in a crystalline state, and above $\text{T}{}_{\mathrm{M}}$, when the alipathic chain are in a fluid state.¹H NMR spectra show that even at the maximum cytochrome $\text{d-b}{}_5$ concentration the presence of the proteins does not markedly change the dynamics to the phospholipid molecules. An asymmetric single-shelled vesicle structure is proposed for the complex.     

The reconstitution of a Photosystem II protein complex, P-700-chlorophyll a-protein complex and light-harvesting chlorophyll ab-protein

A Photosystem II reaction centre protein complex was extracted from spinach chloroplasts using digitonin. This complex showed (i) high rates of dichloroindophenol and ferricyanide reduction in the presence of suitable donors, (ii) low-temperature fluorescence at 685 nm with a variable shoulder at 695 nm which increased as the complex aggregated due to depletion of digitonin and (iii) four major polypeptides of 47, 39, 31 and 6 kDa on dissociating polyacrylamide gels. The Photosystem II protein complex, together woth the P-700-chlorophylla protein complex and light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ complex (LHCP) also isolated using digitonin, were reconstituted with lipids from spinach chloroplasts to form proteoliposomes. The low-temperature (77 K) fluorescence properties of the various proteoliposomes were analysed. The $\text{F}{}_{685}\text{F}{}_{695}$ ratios of the Photosystem II reaction centre protein complex-liposomes decreased as the lipid to protein ratios were increased. The $\text{F}{}_{681}\text{F}{}_{697}$ ratios of LHCP-liposomes were found to behave similarly. Light excitation of chlorophyll b at 475 nm stimulated emission from both the Photosystem II protein complex (F₆₈₅ and F₆₉₅) and the P-700-chlorophyll a-protein complex (F₇₃₅) when LHCP was reconstituted with either of these complexes, demonstrating energy transfer between LHCP and PS I or II complexes in liposomes. No evidence was found for energy transfer from the PS II complex to the P-700-chlorophyll a-protein complex reconstituted in the same proteoliposome preparation. Proteoliposome preparations containing all three chlorophyll-protein complexes showed fluorescence emission at 685, 700 and 735 nm.     

A restatement of melittin-induced effects on the thermotropism of zwitterionic phospholipids

Perturbations induced by melittin on the thermotropism of dimyristoyl-, dipalmitoyl-, distearoylphosphatidylcholine and natural sphingomyelin are investigated and rationalized from data obtained by fluorescence polarization, differential scanning calorimetry and Raman spectroscopy. Depending on the technique and / or experimental conditions used, the observed effects differ at the same lipid to protein molar ratio, due to partial binding of melittin. The binding is more efficient for tetrameric than for monomeric melittin, but in both cases its affinity is weaker for phosphatidylcholine dispersions in the gel phase than for sonicated vesicles. For temperatures $\text{T ? T}{}_{\mathrm{<mtext>m</mtext>}}$ efficient binding occurs whatever the initial state of the lipids is. One can summarize the effects induced by melittin on the transition temperature as follows: (i) No upward shift is observed on synthetic phosphatidylcholines when lipid degradation is avoided. This is achieved by using highly purified melittin, phospholipase inhibitors, and / or non-hydrolysable lipids. (ii) Melittin monomer does not change $\text{T}{}_{\mathrm{<mtext>m</mtext>}}$. (iii) When melittin tetramer is stabilized, it decreases $\text{T}{}_{\mathrm{<mtext>m</mtext>}}$ by 10–15 deg. C. The transition broadens, and is finally abolished for $\text{R}{}_{\mathrm{<mtext>i</mtext>}}\text{? 2}$. Very similar results are found for natural sphingomyelin. Fluorescence polarization indicates similar changes in order and dynamics of the acyl chains for all lipid studied. For $\text{T ? T}{}_{\mathrm{<mtext>m</mtext>}}$, fluorescence and Raman show that melittin decreases the amount of CH₂ groups in ‘trans’ conformation and the intermolecular order of the chains. According to fluorescence data, there is an increase of the rigid-body orientational order at $\text{T ? T}{}_{\mathrm{<mtext>m</mtext>}}$, while from Raman the positional intermolecular order decreases without significant change in the CH₂ groups ‘trans’/‘gauche’ ratio.     

Bacterial bioluminescence-identification of fatty acid as product, its quantum yield and a suggested mechanism

By using radioactive decanal the direct transformation of this aldehyde to decanoic acid, with a quantum yield of 0.13, has been demonstrated. A mechanism analogous to that of other better understood bioluminescent reactions is proposed, leading to a product, as yet unisolated from the enzymic reaction, whose fluorescence spectrum is an excellent match for that of the $\text{in vivo}$ luminescence.The extensive examination^1,2,3 of the isolated bacterial luminescence system has resulted in the accepted outline shown. We wish to modify it, in accordance with the previous evidence, by suggesting that ’intermediates I and II‘ in Hastings' terminology² are the same enzyme bound FMNH₂ moiety.

$\text{FMN}{}_2\text{enzyme}\text{?}\text{enzyme FMNH}{}_2$

$\text{enzyme FMNH}{}_2\text{RCHO}\text{?}\text{covalent complex}$

$\text{covalent complex O}{}_2\text{→}\text{P}{}^1\text{RCO}{}_2\text{H}$

A lively controversy has surrounded the attempts to determine whether aldehyde exerts a purely catalytic role² or is transformed in the reaction.⁴ If the aldehyde reacts, then the simplest product is the corresponding carboxylic acid, perhaps formed via the peracid. The most likely alternative reaction would involve enolistation and oxidation at the α-methylene group. We examined the second alternative fairly carefully, and found no evidence for it. We do not wish to report these results in detail at present, since we have now established that the acid corresponding to that formed in a normal autoxidation of the aldehyde is the product. Some indication of the nature of the products of the reaction is available.⁵Since the amount of product in the reaction is restricted to a very low level by the concentrations required, we labelled decanal with tritium at C-2 and thus were able to record the yield with some precision. Although recent work⁶ strongly implies that acid is formed stoichiometrically, the direct measurement of the quantum yield with respect to acid formation is necessary before a mechanism can be written. We have suggested a mechanism compatible with observations in this system, analogous to all cases of bioluminescence for which a mechanism is reasonably well established. This mechanism also leads to a product excited state with excellent agreement around pH7 in fluorescence wavelength to that of the $\text{in vivo}$ luminescence.     

Transport kinetics of dipicrylamine through lipid bilayer membranes. Effects of membrane structure

Charge-pulse current-relaxation studies have been performed with lipid bilayer membranes in the presence of the hydrophobic ion dipicrylamine. From the analysis of the relaxation times and amplitudes the translocation rate constant $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ of dipicrylamine as well as the partition coefficient β between membrane surface and water could be evaluated. In a first series of experiments membranes made from monoolein or dioleoylphosphatidylcholine in a number of different $\text{n-}\text{alkane}$ solvents were studied, as well as virtually solvent-free bilayer membranes made from monolayers. The thickness $\text{d}$ of the hydrocarbon layer of these membranes varied between 5.0 and 2.5 nm. While β was almost insensitive to variations in $\text{d}$, a strong decrease of $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ with increasing membrane thickness was found; the observed dependence of $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ on $\text{d}$ approximately agreed with the theoretically expected influence of membrane thickness on the height of the dielectric barrier. No specific differences between Mueller-Rudin films and solvent-free (Montal-Mueller) membranes other than differences in thickness were found. In a further series of experiments the chemical structure of the lipid was systematically varied (number and position of double bonds in the hydrocarbon chain, nature of the polar head group). The translocation rate constant $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ was much larger in phosphatidylethanolamine membranes than in phosphatidylcholine membranes. A strong increase of $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ was found when the number of double bonds in the hydrocarbon chain was increased from one to three. These changes were discussed in terms of membrane fluidity and dielectric barrier height. Much higher values of $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ were observed in lipids with ester linkage between hydrocarbon chain and glycerol backbone, as compared with the corresponding ether analogs. This finding is qualitatively consistent with determinations of dipolar potentials in monolayers of ester and ether lipids. When cholesterol is added to phosphatidylcholine membranes, the translocation rate constant $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ increases up to five-fold, while the partition coefficient β remains virtually constant. The variation of $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ in this case can be largely accounted for by a decrease in membrane thickness and a concomitant reduction in dielectric barrier height. In membranes made from the negatively charged lipid phosphatidylserine the partition coefficient of dipicrylamine strongly increased with ionic strength, as expected from the Gouy-Chapman theory of the surface potential.     

6-Phosphogluconolactonase from Zymomonas mobilis: An enzyme of high catalytic efficiency

The enzyme 6-phosphogluconolactonase (EC 3.1.1.31) is present at high levels in Zymomonas mobilis cells. A simple procedure for its isolation involving dye-ligand chromatography and gel filtration has resulted in a 500-fold purification with high recovery. The purified enzyme is a monomer of 26 kDa, and has a high catalytic efficiency with $\text{k}{}_{\mathrm{cat}}\text{K}{}_{\mathrm{m}}$ of 9 × 10⁷ M⁻¹ s⁻¹ at 25° C. Two assay procedures for the enzyme are compared, and a simple method of obtaining a solution of 6-phosphoglucono-δ-lactone relatively free of other metabolites is presented.