Effect of fluorescamine modification of purple membranes on exciton coupling and light-to-dark adaptation

Purple membranes of $\text{Halobacterium}\text{,}\text{halobium}$ were modified with fluorescamine. At pH 8.8, with a molar ratio of fluorescamine to bacteriorhodopsin of 170, about 6 residues of lysine were modified while the arginines were not affected at all. Except for the appearance of the fluorescamine peak at 394 nm and some broadening of the chromophore peak at 570 nm, the absorption spectrum of bacteriorhodopsin was not significantly changed after modification. After fluorescamine modification, circular dichroism studies indicated loss of exciton coupling between bacteriorhodopsin molecules in the purple membrane. Rotational diffusion studies suggested enhanced mobility of the chromophore after modification. However, the spectral changes accompanying the light-to-dark adaptation of purple membranes were not prevented by fluorescamine modification. The implications of these findings are that exciton coupling between neighboring bacteriorhodopsin molecules in the purple membrane is not required for light-to-dark adaptation.     

The transverse location of tryptophan residues in the purple membranes of Halobacterium halobium studied by fluorescence quenching and energy transfer

Fluorescence quenching by a series of spin-labelled fatty acids is used to map the transverse disposition of tryptophan residues in bacteriorhodopsin (the sole protein in the purple membranes of Halobacterium halobium). A new method of data analysis is employed which takes into account differences in the uptake of the quenchers into the membrane. Energy transfer from tryptophan to a set of $\text{n-(9-}\text{anthroyloxy)}$ fatty acids is used as a second technique to confirm the transverse map of tryptophan residues revealed by the quenching experiments. The relative efficiencies of quenching and energy transfer obtained experimentally are compared with those predicted on the basis of current models of bacteriorhodopsin structure. Most of the tryptophan fluorescence is located near the surface of the purple membrane. When the retinal chromophore of bacteriorhodopsin is removed, tryptophan residues deep in the membrane become fluorescent. These results indicate that the deeper residues transfer their energy to retinal in the native membrane. The retinal moiety is therefore located deep within the membrane rather than at the membrane surface.     

Metabolic sequestration of putrescine in Neurospora crassa

The metabolic fate of putrescine labelled $\text{in}\text{vivo}$ was investigated after administration of a trace (10^?7 M) of L-[¹⁴C]ornithine to exponentially growing mycelia of $\text{Neurospora}\text{crassa}$, followed by a large chase (2 mM) of L-[¹²C]ornithine. The specific radioactivities of putrescine and spermidine were determined during the chase period by reaction with [³H]dansyl chloride of known specific radioactivity and isolation of the dansyl-derivatives by thin-layer chromatography. Radioactivity remained in the putrescine pool for over 2 h during the chase period. This suggests that putrescine is largely sequestered (80% or more) $\text{in}\text{vivo}$. The metabolic sequestration of polyamines may be a significant factor in the regulation of polyamine synthesis.     

Photophosphorylation in cell envelope vesicles from Halobacteriumhalobium

We have prepared vesicles from cell envelope membranes of $\text{Halobacterium}\text{halobium}$ strains R₁ and ET-15 which are able to synthesize ATP in response to illumination. This photophosphorylation is inhibited by dicyclohexylcarbodiimide (DCCD) and by phloretin. ATP synthesis in L vesicles from the R₁ strain (which contain bacteriorhodopsin) is inhibited by the protonophore 1799 but not by valinomycin. In M vesicles from the R₁ strain and in ET-15 vesicles (both contain halorhodopsin) photophosphorylation is inhibited by both 1799 and valinomycin. These data are consistent with the idea that light-driven ATP synthesis can be coupled to the electrochemical H⁺ gradient generated by bacteriorhodopsin or by halorhodopsin through the membrane potential component of protonmotive force.     

Improved quantitation of picomolar amounts of valine extracted from muscle cell cultures

A procedure is presented for improving the measurement of picomolar quantities of the amino acid valine extracted from cultured skeletal muscle cells. Data are presented which show (i) the presence of a background ³H contamination resulting from the presence of unreacted 1-[³H]-dimethylaminonaphthalene-5-sulfonyl(dansyl) chloride in the spot of [³H]dansyl [¹⁴C]valine after isolation by micropolyamide thin-layer chromatography, (ii) that even after correction for background ³H contamination, nonequivalent amounts of ³H and ¹⁴C are recovered in the isolated spot, (iii) that the ${}^3\text{H}{}^{14}\text{C}$ recovered is dependent on the amount of dansyl valine applied to the chromatogram, (iv) that the error resulting from background contamination and differential loss of label can be corrected by the use of a standard curve regardless of whether the dansyl valine chromatographed is from a purified solution or a muscle extract, (v) that the presence of varying amounts of other dansyl amino acids in the sample does not affect the validity of the standard curve. This method yields a technique for measuring either picomoles or specific activity which is accurate, reproducible, and highly sensitive.     

Charge asymmetry of the purple membrane measured by uranyl quenching of dansyl fluorescence

Purple membrane was covalently labeled with 5-(dimethylamino) naphthalene-1-sulfonyl hydrazine (dansyl hydrazine) by carbodiimide coupling to the cytoplasmic surface (carboxyl-terminal tail: 0.7 mol/mol bacteriorhodopsin) or by periodate oxidation and dimethylaminoborane reduction at the extracellular surface (glycolipids: 1 mol/mol). In 2 mM acetate buffer, pH 5.6, micromolar concentrations of UO2 +(2) were found to quench the dansyl groups on the cytoplasmic surface (maximum = 26%), while little quenching was observed at the extracellular surface (maximum = 4%). Uranyl ion quenched dansyl hydrazine in free solution at much higher concentrations. Uranyl also bound tightly to unmodified purple membrane, (apparent dissociation constant = 0.8 microM) as measured by a centrifugation assay. The maximum stoichiometry was 10 mol/mol of bacteriorhodopsin, which is close to the amount of phospholipid phosphorus in purple membrane. The results were analyzed on the assumptions that UO2 +(2) binds in a 1:1 complex with phospholipid phosphate and that the dansyl distribution and quenching mechanisms are the same at both surfaces. This indicates a 9:1 ratio of phosphate between the cytoplasmic and extracellular surfaces. Thus, the surface change density of the cytoplasmic side of the membrane is more negative than -0.010 charges/A2.     

Loose binding of testicular mitochondrial ATPase to the inner membrane

Rat testis mitochondrial ATPase was not inhibited by oligomycin at pH 7.5. It was inhibited only at higher alkaline pH's, and showed a lower sensitivity both to oligomycin and N,N′-dicyclohexylcarbodiimide and a higher one to efrapeptin. In submitochondrial particles, testis ATPase was only slightly inhibited by oligomycin, ossamycin, and efrapeptin. The possibility of a loose binding of F₁ to the membrane was supported by its recovery from the supernatant of the submitochondrial particles. Furthermore, by electron microscopy, after hypoosmotic shock and negative staining of the mitochondrial preparations, most of the inner mitochondrial membranes showed only a few “knobs” or none at all. The capacity of the testis mitochondrial preparation to produce ATP was tested and compared to that from liver. ATP synthetase/ATPase activity ratio was $\text{30}\text{1}$ in liver mitochondria, whereas in the testis it was $\text{3}\text{1}$. In spite of this large difference, at least part of the testis ATPase must be firmly bound to the membrane, since it is able to form ATP. The rest seems to be loosely bound and its functional significance is still unknown.     

Determination of the retinal/protein molar ratios for the purple membranes of Halobacterium halobium and Halobacterium cutirubrum

Previously reported values of the retinal/protein molar ratios for the purple membranes from halobacteria are confusing. For $\text{H. halobium}$ values of approximately 1.00 and 0.45 and for $\text{H. cutirubrum}$ values of 0.45 and 0.43 have been published. A redetermination of these ratios in our hands has yielded identical values of 0.99 ± 0.01 for the membranes of both species. This is in agreement with expectations that the ratio be the same for the two species since the similarity of their membrane structure has been established and that every apoprotein molecule be complexed with a retinal since the uniqueness and equivalence of the membrane protein, bacteriorhodopsin, has been demonstrated.     

Fluorescence polarization studies of human and rhesus A-I apolipoproteins and their complexes with phosphatidylcholine.

Human and rhesus A-I apolipoproteins, covalently labelled with dansyl chloride, were used in fluorescence polarization studies of: 1) the monomeric structure of the free proteins in solution; 2) the interaction of the apolipoproteins with sonicated egg phosphatidylcholine; and 3) the size of the saturated complexes of protein with phospholipid. The results indicate that both monomeric apolipoproteins have relatively rigid, yet asymmetrical structures, with Stokes radii of 24.2 ± 0.5 Å, in neutral aqueous solutions. Axial ratios are of the order of $\text{6}\text{1}$ or $\text{4}\text{1}$ for hydrated, prolate or oblate ellipsoids, respectively. A molar excess of about 200 phosphatidylcholine molecules are required to saturate each apolipoprotein. At saturation, the complexes with both proteins have Stokes radii of 40.6 ± 1.7 Å. Since the radius of phosphatidylcholine vesicles is around 125 Å, we conclude that the complexes are relatively small structures derived from disruption of the lipid vesicles, rather than from adsorption of the proteins on intact vesicles.     

Bound cytochrome C as proton donor and acceptor during enzymic oxidoreduction

When ferrocytochrome $\text{c}$ reacts with delipidated cytochrome oxidase under conditions which prevent oxidation, one proton is taken up per molecule of ferrocytochrome $\text{c}$ bound to cytochrome oxidase. When ferricytochrome $\text{c}$ reacts with delipidated Complex III, one proton is released per molecule of ferricytochrome $\text{c}$ bound to Complex III. From these data it can be concluded that the oxidation of ferrocytochrome $\text{c}$ by cytochrome oxidase leads to the release of a proton and an electron, whereas the reduction of ferricytochrome $\text{c}$ by Complex III leads to the uptake of a proton and an electron. Thus ferrocytochrome $\text{c}$ like QH₂ and NADH is both an electron and proton donor, and ferricytochrome $\text{c}$ like Q and O₂ is both an electron and proton acceptor. The pattern for the three mitochondrial electron transfer sequences NADH → Q, QH₂ → ferricytochrome $\text{c}$ and ferrocytochrome $\text{c}$ → O₂ involves separation of an electron and proton on the side of the membrane where electron transfer is initiated and recombination of an electron and a proton in the terminal acceptor on the side of the membrane where electron transfer terminates.     

A new photoaffinity-labeled derivative of mitochondrial cytochrome c

Three lysine residues of horse heart cytochrome $\text{c}$ were modified by reaction with methyl-4-mercaptobutyrimidate hydrochloride and the free SH group of the latter was covalently linked to p-azidophenacyl bromide yielding a photoaffinity-labeled cytochrome $\text{c}$. The photoaffinity-labeled cytochrome $\text{c}$ was bound by irradiation into a covalent complex with cytochrome $\text{c}$ oxidase.     

An improved spectrophotometric assay for human plasma carboxypeptidase N

A continuous spectrophotometric assay for human plasma carboxypeptidase N utilizing furylacryloyl-alanyl-lysine is described. Synthesis was made by use of 9-(2-sulfo)fluorenylmethyloxycarbonyl (Sulfmoc) chloride as the N-?-amino-blocking group for lysine. The substrate has the advantage of containing a chromophore which allows difference measurements above 324 nm. The kinetic parameters K_m and $\text{K}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ have been determined for furylacryloyl-alanyl-lysine and -arginine. Difference measurements were related to micromoles of lysine or arginine released and were expressed as units.     

Synthesis of α-amylase and α-glucosidase by membrane bound ribosomes from Bacilluslicheniformis

α-Glucosidase was membrane bound during exponential growth of $\text{Bacillus}\text{licheniformis}$ but was released into the medium during stationary phase. It could be partially removed from exponential phase cells by washing with NaCl (0.5 M). α-Amylase was exclusively extracellular and could not be detected in cells. Polysomes were prepared from exponential phase cells and separated into membrane bound and soluble fractions. $\text{In}\text{vitro}$ chain completion and immunoprecipitation showed that α-glucosidase and α-amylase were synthesized by membrane bound and not by soluble ribosomes.     

Release of the chloride-dependent arginine aminopeptidase from PMN leukocytes and macrophaces during phagocytosis

The zymosan particles induced a time-dependent release of the chloride-dependent arginine aminopeptidase from rat peritoneal macrophages during $\text{in}$$\text{vitro}$ incubations. Intraperitoneal injections of zymosan, a streptococcal cell preparation and a $\text{Micrococcu}$-suspension caused the release of the chloride-activated arginine aminopeptidase into the peritoneal fluid. The arginine aminopeptidases obtained both from the cell cultivation media and the peritoneal washes were partly purified. The enzymes were similar with regard to the following properties: chloride activation with an optimum at physiological concentrations; strong inhibition by 10^?6M $\text{p}$-chloromercuribenzoate; similar elution properties and preferential hydrolysis of mainly the $\text{N}$-L-aminoacyl-2-naphthylamines of arginine and lysine. The chloride-activated arginine aminopeptidase released into the media in $\text{in}$$\text{vitro}$ conditions was inactivated in contrast to the enzyme released into the peritoneal fluid as a result of the intraperitoneal injections. The timing of the release of the chloride-activated arginine aminopeptidase both $\text{in}$ and $\text{in}$$\text{vitro}$ suggests that the enzyme plays a role in the initial phases of inflammation.     

Studies on (K+ + H+)-ATPase. V. Chemical composition and molecular weight of the catalytic subunit

(1) A $\text{(K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}\text{)-ATPase}$ preparation from porcine gastric mucosa is solubilized in sodium dodecyl sulfate, and is subjected to gel filtration. (2) A main subunit fraction is obtained, which is a protein carbohydrate lipid complex, containing 88% protein, 7% carbohydrate and 5% phospholipid. The detailed composition of the protein and carbohydrate moieties are reported. (3) Sedimentation analysis of the subunit preparation, after detergent removal, reveals no heterogeneity, but the subunits readily undergo aggregation. (4) Acylation of the subunit preparation with citraconic anhydride causes a clear shift of the band obtained after SDS gel electrophoresis, but the absence of broadening and splitting of the band pleads against subunit heterogeneity. (5) Treatment of the subunit preparation with dansyl chloride indicates that the NH₂ terminus is blocked, which favors the assumption of homogeneity of the protein. (6) Binding studies with concanavalin A indicate that at least 86% of the subunit preparation is composed of glycoprotein. (7) These findings, taken together, strongly suggest that there is a single subunit which is a glycoprotein and which represents the catalytic subunit of the enzyme. From sedimentation equilibrium analysis a molecular mass value of 119 kDa (S.E. 3, $\text{n = 6}$) is calculated for protein + carbohydrate and of 110 kDa (S.E. 3, $\text{n = 6}$) for protein only. (8) In combination with the molecular mass of 444 kDa (S.E. 10, $\text{n = 4}$) obtained for the intact enzyme by radiation inactivation we conclude that the enzyme appears to be composed of a homo-tetramer of catalytic subunits.     

Structural properties of the membrane of intact human spermotozoa: A study with fluorescent probes

The properties of the membrane of intact, metabolically active, human persmatozoa have been studied by the use of 1-anilino-8-napthalene sulfonate (ANS). By fluorescence microscopy it was found that at neutral pH ANS is bound exclusively to the membrane of the entire sperm with some preferential binding to the midpiece, while at low pH some preferential binding to the aerosome was observed. By spectrofluorimetry, fluorescence was found to be enhanced 48-fold on binding of ANS to the spermatozoal membrane, with a 50-nm shift in the emission spectrum of the bound dye. $\text{2.47 ± 0.02}$ nmoles of ANS were bound per 10⁶ spermatozoa ($\text{K=2.3–10}{}^{\mathrm{?5}}\text{M}$). Scatchard plots indicate that all the binding sites on the spermatozoal membrane have similar binding characteristics with aZ value of 84.8. Energy transfer with an efficiency of 7% was found for recently ejaculated spermatozoa. The fluorescence of bound ANS depends on the pH of the medium and possibly on the metabolic state of the cell, since addition of succinate or fructose produces an enhancement of fluorescence, while addition of glucose results in a decrease of this parameter. These changes are inhibited by the presence of cyanide.