The biosynthesis of blood group B determinants by the blood group A gene-specified alpha-3-N-acetyl-D-galactosaminyltransferase

The blood group $\text{A}{}^1$ gene-specified α-3-N-acetyl-D-galactosaminyl-transferase in human plasma, when concentrated by adsorption onto group O red cell ghosts or Sepharose 4B, catalyses the transfer of D-galactose in α-linkage to low-molecular-weight H-active acceptors. The product synthesised with 2′-fucosyllactose is chromatographically indistinguishable from the blood group B-active tetrasaccharide, Galα1→3[Fucα1→2]Galβ1→4Glc. The optimum pH for the transfer of D-galactose by the $\text{A}{}^1$-transferase is 7. At this pH the V_max for the transfer of N-acetyl-D-galactosamine is about 300 times higher than that for the transfer of D-galactose. These results indicate that an $\text{A}{}^1$-transferase can, under centain conditions, synthesise B determinant structures.     

Local measurement of lateral motion in erythrocyte membranes by photobleaching technique

The lateral diffusion coefficients ($\text{D}$) of the molecular fluorescence probe 3,3′-dioctadecylindocarbocyanine iodide (DII) in the membrane of discoid erythrocyte ghosts has been measured with the photobleaching technique between 7°C and 40°C. A fluorescence microscope which allows bleaching experiments within small local fields (approx. 1 μm²) at high magnification (X1600) has been used for these measurements. The diffusion coefficient increases from $\text{D = 9 · 10}{}^{\mathrm{?10}}\text{cm}{}^2\text{/s}$ to $\text{D = 7.5 · 10}{}^{\mathrm{?9}}\text{cm}{}^2\text{/s}$ from 7 to 40°C. An increase in membrane fluidity between 12°C and 17°C indicates a conformational change of the lipid bilayer moiety in this temperature region. The diffusion coefficient measured in the regions between the spicules of echinocytes is appreciably smaller than in the untransformed discoid ghosts. In the myelin tubes originating from cells, the lateral diffusion is somewhat larger (about a factor of 2) than in the non-transformed ghosts. With the fluorescence probe technique the rate of growth of myelin tubes of 0.3 μm diameter has been estimated.     

A simple procedure for resolution of Escherichia coli RNA polymerase holoenzyme from core polymerase.

The association constant, K_A, for myosin subfragment-1 binding to actin was measured as a function of ionic strength [KCl, LiCl, and tetramethylammonium chloride (TMAC)]and temperature by the method of time-resolved fluorescence depolarization. The following thermodynamic values were obtained from solutions of 0.20 × 10^?6m S-1, 1.00 × 10^?6m actin in 0.15 m KCl, pH 7.0, at 25 °C: ΔG ° = ?39 ± 1 kJ M^?1, ΔH⁰ = 44 ± 2 kJ M^?1 and $\text{ΔS}{}^0\text{= 0.28 ± 0.01 kJ}\text{M}{}^{\mathrm{?1}}{}^0\text{K}{}^{\mathrm{?1}}$. For measurements in KCl (0.05 to 0.60 m), In $\text{K}{}_{\mathrm{a}}\text{= ?8.36 (KCl)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$. Thus, the binding is endothermic and strongly inhibited by high ionic strength. When KCl was replaced by LiCl or TMAC the ionic effects on the binding were cation specific. The nature of actin-(S-1) binding in the rigor state is discussed in terms of these results.     

The dependence of reaction center and antenna triplets on the redox state of Photosystem I

The formation of chlorophyll triplet states during illumination of Photosystem I reaction center samples depends upon the redox state of P-700, X and ferredoxin Centers A and B. When the reaction centers are in the states P-700⁺A₁XFd_BFd^?_A and P-700 A₁XFd^?_BFd^?_A prior to illumination, we observe electron paramagnetic resonance (EPR) spectra from a triplet species which has zero-field splitting parameters (|D| and |E|) larger than those of either the chlorophyll a or chlorophyll b monomer triplet, and a polarization which results from population of the triplet spin sublevels by an intersystem crossing mechanism. We interpret this triplet as arising from photoexcited chlorophyll antenna species associated with reaction centers in the states P-700⁺Fd^?_A and P-700⁺X^?, respectively, which undergo de-excitation via intersystem crossing. When the reaction centers are in the states P-700A₁XFd^?_BFd^?_A and P-700A₁X^?Fd^?_BFd^?_A prior to illumination, we observe a triplet EPR signal with a polarization which results from population of the triplet spin sublevels by radical pair recombination, and which has a |D| value similar to that of chlorophyll a monomer. We interpret this triplet (the radical pair-polarized triplet) as arising from ³P-700 which has been populated by the process $\text{P-700}{}^{\mathrm{+}}\text{A}{}^{\mathrm{?}}{}_1\text{→}{}^3\text{P-700}\text{A}{}_1$. We observe both the radical pair-polarized triplet and the chlorophyll antenna triplet when the reaction centers are in the state P-700 A₁XFd^?_BFd^?_A, presumably because the processes P-700⁺A^?₁X → P-700⁺A₁X^? and $\text{P-700}{}^{\mathrm{+}}\text{A}{}^{\mathrm{?}}{}_1\text{X}\text{→}{}^3\text{P-700}\text{A}{}_1\text{X}$ have similar rate constants when Centers A and B are reduced, i.e., the forward electron transfer time from A^?₁ to X is apparently much slower in the redox state P-700 A₁XFd^?_BFd^?_A than it is in state P-700 A₁XFd_BFd_A. The amplitude of the radical pair-polarized triplet EPR signal does not decrease in the presence of a 13.5-G-wide EPR signal centered at g 2.0 which was recorded in the dark prior to triplet measurements in samples previously frozen under intense illumination. This g 2.0 signal, which has been attributed to phototrapped A^?₁ (Heathcote, P., Timofeev, K.N. and Evans, M.C.W. (1979) FEBS Lett. 101, 105–109), corresponds to as many as 12 spins per P-700 and can be photogenerated during freezing without causing any apparent attenuation of the radical pair-polarized triplet amplitude. We conclude that species other than A^?₁ contribute to the g 2.0 signal.     

New experimental approach to the estimation of rate of electron transfer from the primary to secondary acceptors in the photosynthetic electron transport chain of purple bacteria

A method for calculating the rate constant ($\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$) for the oxidation of the primary electron acceptor (A₁) by the secondary one (A₂) in the photosynthetic electron transport chain of purple bacteria is proposed.The method is based on the analysis of the dark recovery kinetics of reaction centre bacteriochlorophyll (P) following its oxidation by a short single laser pulse at a high oxidation-reduction potential of the medium. It is shown that in Ectothiorhodospira shaposhnikovii there is little difference in the value of $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ obtained by this method from that measured by the method of Parson ((1969) Biochim. Biophys. Acta 189, 384–396), namely: $\text{(4.5±1.4) · 10}{}^3\text{s}{}^{\mathrm{?1}}$ and $\text{(6.9±1.2) · 10}{}^3\text{s}{}^{\mathrm{?1}}$, respectively.The proposed method has also been used for the estimation of the $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ value in chromatophores of Rhodospirillum rubrum deprived of constitutive electron donors which are capable of reducing P⁺ at a rate exceeding this for the transfer of electron from A₁ to A₂. The method of Parson cannot be used in this case. The value of $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ has been found to be $\text{(2.7±0.8) · 10}{}^3\text{s}{}^{\mathrm{?1}}$.The activation energies for the A₁ to A₂ electron transfer have also been determined. They are 12.4 kcal/mol and 9.9 kcal/mol for E. shaposhnikovii and R. rubrum, respectively.     

Damping of oscillations in the semiquinone absorption in reaction centers after successive flashes. Determination of the equilibrium between QA−QB and QAQB−

A quantitative model for the damping of oscillations of the semiquinone absorption after successive light flashes is presented. It is based on the equilibrium between the states Q_A^?Q_B and Q_AQ_B^?. A fit of the model to the experimental results obtained for reaction centers from Rhodopseudomonas sphaeroides gave a value of $\text{α =}\text{[}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}{}^{\mathrm{?}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}\text{]}\text{([}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}{}^{\mathrm{?}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}\text{] + [}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}{}^{\mathrm{?}}\text{])}\text{= 0.065 ± 0.005}$ (T = 21°C, pH 8).     

The solid state acyl shift of diglycerides: An electron diffraction study

The progress of the solid state acyl shift of 1,2-diglycerides to 1,3-diglycerides is followed at room temperature in single dipalmitin microcrystals by electron diffraction. The β′form, rather than the α-form of the 1,2-isomer, transforms to the 1,3 product. The β′ form packs in the monoclinic paraffin fashion, i.e. the O_⊥ methylene subcell and a chain tilt of 27° about the long subcell axis. After the isomerization, the chain tilt (14° to surface normal) occurs around the $\text{b}{}_{\mathrm{<mtext>S</mtext>}}{}^1$ axis of the resultant T_∥ methylene subcell.     

Membrane lipid dynamics and enzymic activity in bovine adrenal cortex microsomes

The lipid dynamics of the adrenocortical microsomal membranes was studied by monitoring the fluorescence anisotropy and excited state lifetime of a set of anthroyloxy fatty acid probes (2-, 7-, 9- and 12-(9-anthroyloxy)-stearic acid (AP) and 16-(9-anthroyloxy)palmitic acid (AS). It was found that a decreasing polarity gradient from the aqueous membrane interface to the membrane interior, was present. This gradient was not modified by the proteins, as evidenced by comparison of complete membranes and derived liposomes, suggesting that the anthroyloxy probes were not in close contact with the proteins. An important change of the value of the mean rotational relaxation time as a function of the position of the anthroyl ring along the acyl chain was evidenced. In the complete membranes, a relatively more fluid medium was evidenced in the C₁₆ as compared to the C₂ region, while the rotational motion appeared to be the most hindered at the C₇–C₉ level. In the derived liposomes, a similar trend was observed but the mobility was higher at all levels. The decrease of the mean rotational relaxation time was more important for 12-AS and 16-AP. Temperature dependence of the mean rotational relaxation time of 2-AS, 12-AS and 16-AP in the complete membranes revealed the existence of a lipid reorganization occurring around 27°C and concerning mainly the C₁₆ region. The extent to which the acyl chain reacted to this perturbation at the C₁₂ level depended on pH. The presence of proteins increased the apparent magnitude of this reorganization and also modified the critical temperature from approx. 23°C in the derived liposomes to approx. 27°C in the complete membranes. Thermal dependence of the maximum velocity of the $\text{3-}\text{oxosteroid}\text{Δ}{}^5\text{?Δ}{}^4\text{-}\text{isomerase}$, the second enzyme in the enzymatic sequence, responsible for the biosynthesis of the $\text{3-}\text{oxo}\text{-Δ}{}^4\text{-}\text{steroids}$ in the adrenal cortex microsomes, was studied. The activation energy of the catalyzed reaction was found to be low and constant ($\text{2–5}\text{kcal}\text{·}\text{mol}{}^{\mathrm{?1}}$) in the temperature range 16–40°C at pH 7.5, 8.5 and 9, corresponding to the minimum, intermediate and maximum rate, respectively. A drastic increase of the activation energy ($\text{20}\text{kcal}\text{·}\text{mol}{}^{\mathrm{?1}}$) was observed at temperature below 16°C at pH 7.5. A correlated change of the $\text{p}\text{K}{}_{\mathrm{<mtext>ES</mtext>}}{}^{\mathrm{<mtext>app</mtext>}}$ as function of temperature was detected; at 36°C $\text{p}\text{K}{}_{\mathrm{<mtext>ES</mtext>}}{}^{\mathrm{<mtext>app</mtext>}}\text{= 8.3}$ while at 13°C the value shifted to 8.7. The pH range of the group ionization was narrower at 13°C. In contrast with the behaviour of the $\text{3β-}\text{hydroxy}\text{-Δ}{}^5\text{-}\text{steroid dehydrogenase}$, the $\text{3-}\text{oxosteroid}\text{Δ}{}^5\text{?Δ}{}^4\text{-}\text{isomerase}$ was apparently unaffected by the lipid reorganization at 27°C. It is suggested that this enzyme possesses a different and more fluid lipid environment than the bulk lipids.     

The solvent contribution to the free energy of protein--ligand interactions.

The intrinsic solvent contribution to the free energy of protein-ligand interactions in solution is shown to be related to a free energy per unit area term, obtained from analysis of the solution to gas phase process, and the change in accessible area on association. Analysis of the free energy data on a per unit area basis for the solution to gas phase process leads to the conclusion that the aliphatic CH₂ group is only slightly intrinsically hydrophobic, $\text{δΔG°/}\text{A}\text{?}{}^2\text{= 6}\text{cal mol}{}^{\mathrm{?1}}\text{A}\text{?}{}^2$, whereas the aromatic compound are actually intrinsically hydrophilic, $\text{δΔG°/}\text{A}\text{?}{}^2\text{= -26}\text{cal mol}{}^{\mathrm{?1}}\text{A}\text{?}{}^2$. This leads to the conclusion that, for the interaction of benzene, naphthalene and anthracene with the binding site of α-chymotrypsin, the ligand-solvent free energy contribution is actually unfavorable. Since the protein-solvent contribution is small or unfavorable, the central conclusion is that the solvent contribution to protein-ligand interactions is small or unfavorable and that it is the protein-ligand non-bonded interactions that provide the driving force for association.     

Effects of insulin on the lipid structure of liver plasma membrane measured with fluorescence and ESR spectroscopic methods

Insulin increased the lipid order of rat and mouse liver plasma membrane domains sampled by the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene in a concentration-dependent saturable manner. The ordering is half maximal at $\text{5.1 · 10}{}^{\mathrm{?11}}\text{M}$ and fully saturated at $\text{1.7 · 10}{}^{\mathrm{?10}}\text{M}$ insulin. Membranes prepared from obese hyperglycemic (ob / ob) mice demonstrated a right-shift in the dose-dependent ordering induced by insulin, such that ordering was half maximal at $\text{1.2 · 10}{}^{\mathrm{?10}}\text{M}$ and fully saturated at $\text{2.0 · 10}{}^{\mathrm{?10}}\text{M}$. Insulin also increased the order of rat liver plasma membranes labeled with the cis- and trans-parinaric acid methyl esters. The ordering caused by insulin as detected with cis methyl parinarate was complete within approx. 15 min. after hormone addition at 37°C, and the ordering was approximately double that observed with the trans isomer. Additional ESR experiments demonstrated that the addition of insulin increased the outer hyperfine splittings of spectra recorded from membranes labeled with the steroid-like spin labels, nitroxide cholestane and nitroxide androstane, but not the fatty acid spin probe, 5-nitroxide stearate. Studies utilizing model membrane systems strongly suggest that the 5-nitroxide stearate samples a cholesterol-poor domain of the membrane, while the steroid-like probes preferentially sample cholesterol-rich regions of the membrane. Finally, insulin-induced membrane ordering was dose-dependently inhibited by cytochalasin B in the range 1–50 μM. From these results, we conclude that (1) the ordering effect of insulin addition to isolated liver plasma membrane fractions occurs within the physiological range of hormone concentration, and the dose-response is right-shifted in membranes from ‘insulin resistant’ animals; (2) the relative responses of the fluorescent and spin probes suggest that the effects of insulin are confined to specific domains within the membrane matrix; and (3) the direct effects of insulin on the membranes may involve protein components having cytochalasin B binding sites.     

Acid dissociation of cyclohexaamylose and cycloheptaamylose

Acid dissociation constants of aqueous cyclohexaamylose (6-Cy) and cycloheptaamylose (7-Cy) have been determined at 10–47 and 25–55°C, respectively, by pH potentiometry. Standard enthalpies and entropies of dissociation derived from the temperature dependences of these pK_a's are ΔH⁰ = 8.4 ± 0.3 kcal mol^?1, $\text{Δ}\text{S}{}^0\text{= ?28. ± 1}\text{cal mol}{}^{\mathrm{?1}}{}^0\text{K}{}^{\mathrm{?1}}$ for 6-Cy and ΔH⁰ = 10.0 ± 0.1 kcal mol^?1, $\text{Δ}\text{S}{}^0\text{= ?22.4 ±0.3}\text{cal mol}{}^{\mathrm{?1}}{}^0\text{K}{}^{\mathrm{?1}}$ for 7-Cy. Intrinsic ¹³C nmr resonance displacements of anionic 6- and 7-Cy were measured at 30°C in 5% D₂O ($\text{v}\text{v}$). These results indicate that the dissociation of 6- and 7-Cy involves both C2 and C3 2⁰-hydroxyl groups. The thermodynamic and nmr parameters are discussed in terms of interglucosyl hydrogen bonding.     

Photosystem II energy coupling in chloroplasts with H2O2 as electron donor

NH₂OH-treated, non-water-splitting chloroplasts can oxidize H₂O₂ to O₂ through Photosystem II at substantial rates (100–250 μequiv · h^?1 · mg^?1 chlorophyll with 5 mM H₂O₂) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H₂O₂ → Photosystem II → dimethylquinone reaction supports phosphorylation with a $\text{P}\text{e}{}_2$ ratio of 0.25–0.35 and proton uptake with $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.67 (pH 8)–0.85 (pH 6). These are close to the $\text{P}\text{e}{}_2$ value of 0.3–0.38 and the $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.7–0.93 found in parallel experiments for the H₂O → Photosystem II → dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor → Photosystem II → dibromothymoquinone (→O₂) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I^?, ferrocyanide).     

Temperature dependence of N-phenyl-1-naphthylamine binding in egg lecithin vesicles

The temperature dependence of the binding of PhNapNH₂ ($\text{N-}\text{phenyl-1-naphthylamine}$) to vesicles of egg phosphatidylcholine has been determined. The Arrhenius plot of the association constant exhibits a discontinuity at 20.9 °C, some 30 °C above the broad phase transition region of the phospholipid. In the temperature range above 20 °C, $\text{ΔH}{}^0\text{= ?6100}\text{cal·mol}{}^{\mathrm{?1}}$ and $\text{ΔS}{}^0\text{= 9.7}\text{e. u.}$; in the temperature range below 20 °C, $\text{ΔH}{}^0\text{= 0}\text{cal · mol}{}^{\mathrm{?1}}$ and $\text{ΔS}{}^0\text{= 30.4}\text{e. u.}$ These values are consistent with the view that there are well ordered lipid-lipid bonds below 20 °C which are significantly less important above this temperature. The order in the temperature range of 5 to 20 °C, though significantly greater than that above 20 °C, is still significantly less than that in the crystalline state.     

Kinetics of phospholipid exchange between bilayer membranes

In an accompanying publication by Duckwitz-Peterlein, Eilenberger and Overath ((1977) Biochim. Biophys. Acta 469, 311–325) it is shown that the exchange of lipid molecules between negatively charged vesicles consisting of total phospholipid extracts from Escherichia coli occurs by the transfer of single lipid monomers or small micelles through the water. Here a kinetic interpretation is presented in terms of a rate constant, $\text{k}{}_{\mathrm{?}}$, for the escape of lipid molecules from the vesicle bilayer into the water. The evaluated rate constants are $\text{k}{}_{\mathrm{?}}{}^{\mathrm{<mtext>P</mtext>}}\text{= (0.86 ± 0.05) · 10}{}^{\mathrm{?5}}\text{s}{}^{\mathrm{?1}}$ and $\text{k}{}_{\mathrm{?}}{}^{\mathrm{<mtext>E</mtext>}}\text{= (1.09 ± 0.13) · 10}{}^{\mathrm{?6}}\text{s}{}^{\mathrm{?1}}$ for phospholipid molecules with $\text{trans-}\text{Δ}{}^9\text{-hexadecenoate}$ and $\text{trans-}\text{Δ}{}^9\text{-octadecenoate}$, respectively, as the predominant acyl chain component. The rate constants are discussed in terms of the acyl chain and polar head group composition of the lipids.     

Characterization of beta-adrenergic receptor and adenylate cyclase in canine cerebellum.

Binding of (?)-[${}^3\text{H}$]dihydroalprenolol to the synaptic membrane fractions of canine cerebellum was rapid and reversible with rate constants of 1.62 × 10⁸m^?1 min^?1 and 0.189 min^?1 for the forward and reverse reactions, respectively. The binding was of high affinity and saturable with an equilibrium dissociation constant (K_D) of 5 to 7 nm. Bound (?)-[${}^3\text{H}$]-dihydroalprenolol was displaceable with β-adrenergic agonists and antagonists, but not with a variety of other neuroactive substances such as acetylcholine, histamine, serotonin, dopamine, tyramine, (?)-phenylephrine, γ-aminobutyric acid, glycine, and glutamic acid. Adenylate cyclase of the membranes was stimulated at most three times by β-adrenergic agonists, but not significantly by the other neuroactive substances. Guanine nucleotides such as GTP and guanyl-5′-yl imidodiphosphate (Gpp(NH)p) were strictly required for β-adrenergic stimulation of adenylate cyclase with their optimum concentrations of 50 μm, although the nucleotides alone elevated virtually no basal activity. The affinities of β-adrenergic ligands including some stereoisomers for (?)-[${}^3\text{H}$]dihydroalprenolol binding sites were very similar to those for adenylate cyclase in the presence of GTP. Binding of β-adrenergic agonists to the membranes exhibited an apparent negative cooperativity as determined by displacement of (?)-[${}^3\text{H}$]dihydroalprenolol in the absence of purine nucleotides. This negative cooperativity was entirely abolished by addition of either GTP or Gpp(NH)p at 50 μm. Both (?)-isoproterenol-stimulated adenylate cyclase activity and binding of (?)-[${}^3\text{H}$]dihydroalprenolol were not affected by β₁-selective antagonists, (±)-atenolol, and (±)-practolol, at concentrations which completely inhibit peripheral β₁-responses in vitro, whereas β₂-selective agonists such as YM-08316 (BD-40A) and (±)-salbutamol not only stimulated adenylate cyclase but also competitively inhibited binding of (?)-[${}^3\text{H}$]dihydroalprenolol. These results indicate that canine cerebellar adenylate cyclase may be coupled specifically with β₂-adrenergic receptor.     

Ultracentrifugation studies on early stage polymerization of tobacco mosaic virus protein

The effects of absolute temperature (T), ionic strength (μ), and pH on the polymerization of tobacco mosaic virus protein from the 4 S form (A) to the 20 S form (D) were investigated by the method of sedimentation velocity. The loading concentration in grams per liter (C) was determined at which a just-detectable concentration (β) of 20 S material appeared. It was demonstrated experimentally that under the conditions employed herein, an equilibrium concentration of 20 S material was achieved in 3 h at the temperature of the experiment and that 20 S material dissociated again in 4 h or less to 4 S material either upon lowering the temperature or upon dilution. Thus, the use of thermodynamic equations for equilibrium processes was shown to be valid. The equation used to interpret the results, log ($\text{C?β) =}\text{constant}\text{+ (}\text{ΔH}{}^1\text{2.3RT}$) + ($\text{Δ}\text{W}{}^1{}_{\mathrm{<mtext>el</mtext>}}\text{2.3RT}$) ? K′_sμ + ζpH, was derived from three separate models of the process, the only difference being in the anatomy of the constant; thus, the method of analysis is essentially independent of the model. $\text{ΔH}{}^1$ and ΔW¹_el are the enthalpy and the change in electrical work per mole of A protein (the trimer of the polypeptide chain), K^′_s is the salting-out constant on the ionic strength basis, ζ is the number of moles of hydrogen ion bound per mole of A protein in the polymerization, and R is the gas constant. The three models leading to this equation are: a simple 11th-order equilibrium between A₁ (the trimer of the polypeptide chain) and D, either the double disk or the double spiral of approximately the same molecular weight, designated model A; a second model, designated B, in which A₁ was assumed to be in equilibrium with D at the same time that it is in equilibrium with A₂, A₃, etc., dimers and trimers, etc., of A₁ in an isodesmic system; and a phase-separation model, designated model C, in which A protein is treated as a soluble material in equilibrium with D, considered as an insoluble phase. From electrical work theory, $\text{Δ}\text{W}{}_{\mathrm{el}}{}^1\text{/T}$ was shown to be essentially independent of T; therefore, in experiments at constant μ and constant pH the equation of log (C ? β) versus 1/T is linear with a slope of $\text{ΔH}{}^1\text{/2.3R}$. The results fit such an equation over nearly a 20 °C-temperature range with a single value of $\text{Δ}\text{H}{}^1$ of +32 kcal/mol A₁. Results obtained when T and pH were held constant but μ was varied did not fit a straight line, which shows that more than simple salting-out is involved. When the effect of ionic strength on the electrical work contribution was considered in addition to salting-out, the data were interpreted to indicate a value of $\text{Δ}\text{W}{}^1{}_{\mathrm{el}}$ of 1.22 kcal/mol A₁ at pH 6.7 and a value of 4.93 for K_s^′. When μ and T were held constant but pH was varied, and when allowance was made for the effect of pH changes on the electrical work contribution, a value of 1.1 was found for ζ. This means that something like 1.1 mol of hydrogen ion must be bound per mole of A₁ protein in the formation of D. When this is added to the small amount of hydrogen ion bound per A₁ before polymerization, at the pH values used, it turned out that for D to be formed, 1.5 H⁺ ions must be bound per A₁ or 0.5 per protein polypeptide chain. This amounts to 1 H⁺ ion per polypeptide chain for half of the protein units, presumably those in one but not the other layer of the double disk or turn of the double spiral. When polymerization goes beyond the D stage, as shown by previously published data, additional H⁺ ions are bound. Simultaneous osmotic pressure studies and sedimentation studies were carried out, in both cases as a function of loading concentration C. These results were in complete disagreement with models A and C but agreed reasonably well with model B. The sedimentation studies permitted evaluation of the constant, β, to be 0.33 g/liter.     

Crystal data for tonin, an enzyme involved in the formation of angiotensin II.

Tonin is an enzyme isolated from the submaxillary glands of old rats. This enzyme is involved in the direct conversion of angiotensinogen or the tetradecapeptide renin substrate to the octapeptide angiotensin II, the agent implicated in hypertension. Large well-formed crystals of tonin were grown from 2.5 m-(NH₄)₂SO₄, 0.01 m-phosphate buffer (pH 6.2) and 1% methylpentanediol. The crystals are trigonal, space group P3₁21 (P3₂21) with unit cell dimensions: $\text{a = b = 94}\text{A}\text{?}\text{, c = 66}\text{A}\text{?}\text{, γ = 120°}$. The value of $\text{V}{}_{\mathrm{<mtext>m</mtext>}}\text{= 2.6}\text{A}\text{?}{}^3\text{/}\text{dalton}$ corresponds to one molecule of molecular weight 32,000 per asymmetric unit. Definitive classification of tonin into one of the four proteolytic enzyme classes (carboxyl protease, serine protease, thiol protease or metalloprotease) has not been firmly established.     

Occurrence of passive furosemide-sensitive transmembrane potassium transport in cultured cells

Furosemide ($\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) inhibits a proportion of the total passive (ouabain-insensitive) K⁺ influx into primary chick heart cell cultures (85%), BC₃H1 cells (75%), MDCK cells (40%) and HeLa cells (57%). This action of furosemide upon K⁺ influx is independent of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{pump}$ inhibition since the furosemide-sensitive component of the K⁺ influx is identical in the presence and absence of ouabain ($\text{1 · 10}{}^{\mathrm{?3}}\text{M}$). For HeLa cells the passive, furosemide-sensitive component of K⁺ influx is markedly dependent upon the external K⁺, Na⁺ and Cl^? content. Acetate, iodide and nitrate are ineffective as substitutes for Cl^?, whereas Br^? is partially effective. Partial Cl^? replacement by NO₃^? gave an apparent affinity of 100 mM [Cl]. Na⁺ replacement by choline⁺ abolishes the furosemide-sensitive component, whereas Li⁺ replacement reduces this component by 48%. Partial Na⁺ replacement by choline⁺ gives an apparent affinity of 25 mM [Na⁺]. Variation in the external K⁺ content gives an affinity for the furosemide-sensitive component of approx. 1.0 mM. Furosemide inhibition of the passive K⁺ inflúx is of high affinity, half-maximal inhibition being observed at $\text{5 · 10}{}^{\mathrm{?6}}\text{M}$ furosemide. Piretanide ($\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) and phloretin ($\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) inhibit the same component of passive K⁺ influx as furosemide; ethacrynic acid and amiloride ($\text{both}\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) partially so. The stilbene, SITS ($\text{1 · 10}{}^{\mathrm{?6}}\text{M}$), was ineffective as an inhibitor of the furosemide-sensitive component.     

Some observations on a new type of point average molecular weight

A new type of (reduced) point average molecular weight A¹, is described. Several interesting properties are developed: (i) $\text{A}{}^1\text{=}\text{reduced}$ weight average molecular weight over the whole cell, $\text{A}{}_{\mathrm{<mtext>w</mtext>}}{}^{\mathrm{o}}\text{A}{}^1\text{(}\text{meniscus}\text{) = A}{}_{\mathrm{<mtext>w</mtext>}}\text{(}\text{meniscus}\text{); (iii) A}{}^1\text{(}\text{zero concentration}\text{) =}\text{reduced}$ number average molecular weight, A_n (meniscus). In addition, its usefulness in extracting the meniscus concentration, J(a), and in examining heterogeneous systems such as mucus glycoproteins, are discussed. The evaluation and application of $\text{A}{}^1$ requires only simple computational facilities, without the use for large-scale multiple data acquisition and recycling techniques.