A one-step procedure for the isolation of fructose 1,6-bisphosphatase and fructose 1,6-bisphosphate aldolase from rabbit liver

Human ceruloplasmin, which is usually cleaved by limited proteolysis into three major fragments during preparation ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{? 18,650}$, 50,000, and 70,000) was isolated in good yield as an undegraded single-chain protein ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{? 135,00}$). The cryosupernatant from fresh frozen plasma (100 liters) was fractionated with polyethylene glycol (PEG 4000) at + 5°C yielding a ceruloplasmin-enriched fraction in the 20% PEG supernatant. Three steps of chromatography on DEAE-Sephacel, hydroxyapatite, and Sephadex G-200 produced a homogeneous protein with maximal enzymatic activity and the $\text{A}{}_{610}\text{A}{}_{280}$ ratio of 0.046 corresponding to 98–100% purity. Two forms of ceruloplasmin having this absorbance ratio were obtained; Form I was predominant and was studied further. The procedure separated both forms from apoceruloplasmin and degraded ceruloplasmin. The single-chain ceruloplasmin (Form I) had an NH₂-terminal sequence of Lys-Glu-Lys-His-Tyr-Tyr-Ile-, the same as for the 70,000 fragment, and is suitable for structural study by sequence analysis and physicochemical methods.     

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.     

Synthesis and characterization of a DNA probe complementary to rat liver 28S ribosomal RNA.

Conditions for the production of a complementary DNA sequence for use in studies of ribosomal RNA are described. $\text{E}$. $\text{coli}$ DNA polymerase I is used to transcribe highly purified 28S ribosomal RNA from rat liver. The reaction is sensitive to the tertiary structure of the rRNA template-primer. The complementary DNA hybridizes to its rRNA template with a $\text{R}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 0.02. The hybrid formed between 28S ribosomal RNA and complementary DNA has a T_m of 73°C. The probe reacts with total rat nuclear RNA with a $\text{R}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 1.0.     

Cytoplasmic DNA: Differentiation by reassociation kinetics

Rat liver nuclear and cytoplasmic DNA samples were denatured and the kinetics of their reassociation was measured. About 85% of the soluble cytoplasmic (mitochondrial) DNA reannealed rapidly with a $\text{C}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 0.03}$ while 65% of the particulate (microsomal) DNA reassociated with a $\text{C}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 0.14}$ Both nucleic acids were clearly differentiated from nuclear DNA in their reassociation kinetics. The results indicate that both mitochondrial and microsomal DNA consist mainly of single components or closely related families with repetitive sequences.     

Preferential solvation of methylmercurated calf thymus deoxyribonucleic acid.

The changes in polymer-solvent interactions that occur when native calf thymus DNA is dialyzed against Na₂SO₄ solutions of a given ionic strength and buffer concentration but of varying concentrations in methylmercuric hydroxide have been investigated with the help of solution density measurements at 25 °C and pH 6.8–7.0. From measurements executed under equilibrium dialysis conditions at the three salt levels 5 mm, 0.05 m, and 0.5 m Na₂SO₄ (m refers to molality) and in the presence of 5 mm cacodylic acid buffer, the density increments $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ for native calf thymus DNA were determined as a function of CH₃HgOH concentration. $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ was found not to vary with organomercurial concentration, irrespective of the concentration of supporting electrolyte, until a certain CH₃HgOH concentration level has been reached, viz., , beyond which $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ increases strongly with increasing concentration of CH₃HgOH. As is shown by optical melting, $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ becomes a function of organomercurial concentration the moment DNA undergoes denaturation brought about by the complexing of CH₃HgOH with the various N-binding sites of the base residues in the DNA double helix.Polymer-solvent interactions, expressed in terms of preferential water interactions (“net hydration”) and preferential salt interactions (“salt solvation”), were derived from the $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ data in combination with data obtained on the preferential interaction of CH₃HgOH with denatured DNA and data on the partial specific volumes of all major solution components, gathered from density measurements on solutions with fixed concentrations of diffusible components. Evidence is presented which shows that denaturation in general decreases the net hydration while salt becomes preferentially associated with the polyelectrolyte. This process is further amplified by the interaction of CH₃HgOH with denatured DNA: Methylmercurated DNA alters the redistribution of diffusible components at dialysis equilibrium to such an extent that in a formal sense large amounts of water are rejected from the immediate vicinity of the polymer. The molecular implications of these findings are explored. The results are further discussed in the light of previous findings where the methylmercury-induced denaturation of DNA had been studied with the help of buoyant density measurements in a Cs₂SO₄ density gradient and by velocity-sedimentation in a variety of sulfate media.     

Bicarbonate ion-ATPase in rat liver cell fractions

The distribution of $\text{HCO}{}_3{}^{\mathrm{?}}\text{-}\text{ATPase}$ activity was studied in cell fractions prepared from homogenates of rat liver. The level of mitochondrial contamination in the microsomal fraction depended on the fractionation procedure and on the method of homogenization. With proper care, microsomes with undetectable mitochondrial contamination could be prepared. These microsomes had no detectable $\text{HCO}{}_3{}^{\mathrm{?}}\text{-ATPase}$ activity. Approximately 85 % of the total $\text{HCO}{}_3{}^{\mathrm{?}}\text{-ATPase}$ activity of the post 6000 x g · min supernatant was recovered in the mitochondrialfraction. The properties of this mitochondrial $\text{HCO}{}_3{}^{\mathrm{?}}\text{-}\text{ATPase}$ were not distinguishable from those of the various microsomal $\text{HCO}{}_3{}^{\mathrm{?}}\text{-}\text{ATPase}$ previously described by other investigators.     

Catalytic function of thymidylate synthase is confined to S phase due to its association with replitase

Catalytic activity of thymidylate synthase, as measured $\text{in}\text{,}\text{vivo}$, is tightly linked to S phase of the cell cycle in Chinese hamster embryo fibroblast cells. This activity, as measured $\text{in}\text{,}\text{vitro}$, is found in all parts of the cell cycle. Thymidylate synthase activity in nuclear (karyoplast) extracts increased as the cells progressed from $\text{G}{}_0\text{G}{}_1$ to S phase. This enzymatic activity in the nuclei of S phase cells is associated with the multienzyme complex (replitase) that also contained DNA polymerase and other enzymes of DNA replication and precursor synthesis. The degree of association of thymidylate synthase with replitase, which increased co-ordinately as the cells progressed from $\text{G}{}_0\text{G}{}_1$ phase to S phase, coincided strongly with the level of $\text{in}\text{,}\text{vivo}$ activity of the enzyme.     

Anaerobic stimulation of sugar transport in avian erythrocytes

The kinetic parameters of the sugar transport in avian erythrocytes were evaluated under aerobic and anaerobic conditions. In anaerobic cells, transport measurements with $\text{3-O-[}{}^{14}\text{C}\text{]}$ methylglucose resulted in a set of similar dissociation-like constants. Thus the Michaelis constants of $\text{3-O-[}{}^{14}\text{C}\text{]}$ methylglucose entry and exit, $\text{K}{}_{\mathrm{<mtext>so</mtext>}}$ and $\text{K}{}_{\mathrm{<mtext>si</mtext>}}$, were 8 and 7 mM, respectively. The equilibrium exchange constant, $\text{B}{}_{\mathrm{<mtext>s</mtext>}}$, and the counterflow constant, $\text{R}{}_{\mathrm{<mtext>s</mtext>}}$, were 9 and 11 mM, respectively. The activity constant for $\text{3-O-}\text{methylglucose}$ transport, $\text{F}{}_{\mathrm{<mtext>s</mtext>}}$, defined as $\text{V/K}{}_{\mathrm{<mtext>m</mtext>}}$, was 4 ml/h per g. This set of kinetic constants was compatible with a symmetrical mobile-carrier model. In contrast, the Michaelis constant for glucose entry, $\text{K}{}_{\mathrm{<mtext>go</mtext>}}$, was 2 mM and less than the counterflow constant, $\text{R}{}_{\mathrm{<mtext>g</mtext>}}$ (8 mM). This result could be accounted for by slower movement of the glucose-carrier complex than the free carrier. The activity constant for glucose transport, $\text{F}{}_{\mathrm{<mtext>g</mtext>}}$, was 5 ml/h perg.Under aerobic conditions, two of the dissociation-like constants ($\text{K}{}_{\mathrm{<mtext>si</mtext>}}$ and $\text{B}{}_{\mathrm{<mtext>s</mtext>}}$) for $\text{3-O-}\text{methylglucose}$ transport were significantly larger than those obtained in anaerobic cells, but the remaining two ($\text{K}{}_{\mathrm{<mtext>so</mtext>}}$ and $\text{R}{}_{\mathrm{<mtext>s</mtext>}}$) remained unchanged. The values, for $\text{K}{}_{\mathrm{<mtext>so</mtext>}}\text{, K}{}_{\mathrm{<mtext>si</mtext>}}\text{, B}{}_{\mathrm{<mtext>s</mtext><mtext>\; and</mtext>}}\text{R}{}_{\mathrm{<mtext>s</mtext>}}$ were 8, 26, 20 and 8 mM, respectively. The activity constant, $\text{F}{}_{\mathrm{<mtext>s</mtext>}}$, decreased to 2 ml/h per g. These changes in kinetic constants were consistent with the hypothesis that anoxia accelerated sugar transport by releasing free carrier that was previously sequestered on the inside of the cell membrane.     

Influence of ionic strength upon the proton inventory for the water-catalyzed hydrolysis of N-acetylbenzotriazole

Proton inventory investigations of the hydrolysis N-acetylbenzotriazole at pH 3.0 (or the equivalent point on the pD rate profile) have been conducted at two different temperatures and at ionic strengths ranging from 0 to 3.0 M. The solvent deuterium isotope effects and proton inventories are remarkably similar over this wide range of conditions. The proton inventories suggest a cyclic transition state involving four protons contributing to the solvent deuterium isotope effect for the water-catalyzed hydrolysis. The hydrolysis data are described by the equation $\text{k}{}_{\mathrm{n}}\text{= k}{}_{\mathrm{<mtext>o</mtext>}}\text{(1 ? n + nπ}{}_{\mathrm{<mtext>a</mtext>}}{}^1\text{)}{}^4$ with $\text{π}{}_{\mathrm{<mtext>a</mtext>}}{}^1\text{～ 0.74}$, where k_o is the observed first-order rate constant in protium oxide, n is the atom fraction of deuterium in the solvent, k_n is the rate constant in a protium oxide-deuterium oxide mixture, and $\text{π}{}_{\mathrm{<mtext>a</mtext>}}{}^1$ is the isotopic fractionation factor.     

Cytoplasmic poly(A) polymerase from sea urchin eggs, merogons, and embryos

The presence of cytoplasmic poly(A) polymerase has been established in sea urchin eggs and four-cell embryos by subcellular fractionation and use of enucleate egg halves. ATP is the only ribonucleoside triphosphate incorporated. This incorporation is time dependent, contingent on input protein concentration, and immune to a variety of antimetabolites known to inhibit DNA-directed RNA synthesis. Both the unfertilized egg and the four-cell embryo cytoplasmic poly(A) polymerase activities display a preference for Mn²⁺. While oligo(A)₄ is inactive as a primer, addition of $\text{oligo(A)}{}_{\mathrm{<mtext>16</mtext>}}\text{,}\text{poly(A)}{}_{\mathrm{<mtext>45</mtext>}}$ and $\text{poly(A)}{}_{\mathrm{<mtext>90</mtext>}}$ stimulates ATP incorporation. On a unit per milligram protein basis, the endogenous activity associated with cytoplasmic fractions obtained from nucleate and enucleate egg halves is 36 and 83% that obtained with the cytoplasmic fraction prepared from the unfertilized egg. In the presence of $\text{oligo(A)}{}_{\mathrm{<mtext>16</mtext>}}$, both the nucleate and enucleate egg halves exhibit 81% of the activity associated with the unfertilized egg cytoplasmic fraction. The level of Mn²⁺ cytoplasmic poly(A) polymerase activity from the four-cell embryo is approximately 50% that of the unfertilized egg. This decrease does not appear to be due to either a postfertilization alteration in the subcellular localization of poly(A) polymerase or an increase in RNase activity. Supplementation with $\text{oligo(A)}{}_{\mathrm{<mtext>16</mtext>}}$ failed to restore the four-cell embryo cytoplasmic poly(A) polymerase potential to a level comparable to that of the unfertilized egg. Suppression of postfertilization protein synthesis by emetine, however, prevents this developmental decline in ATP incorporation thereby suggesting that postfertilization cytoplasmic poly(A) polymerase activity is subject to negative translational control.     

Involvement of the proton electrochemical gradient in genetic transformation in Escherichia coli

Plasmid pIY2 DNA which encodes for ampicillin-resistance was used to study the energetics of Ca⁺⁺-induced transformation in Escherichia coli. When cells are exposed to DNA in the presence of carbonylcyanide-m-chlorophenylhydrazone or 2,4-dinitrophenol, two protonophores that collapse the proton electrochemical gradient across the cell membrane ($\text{Δ}\text{μ}{}_{\mathrm{H}}\text{+}$), transformation to ampicillin-resistance is drastically reduced with little or no effect on viability. Furthermore, when the components of $\text{Δ}\text{μ}{}_{\mathrm{H}}\text{+}$ are altered by varying ambient pH or by performing transformation in the presence of valinomycin or nigericin, the efficiency of transformation is directly correlated with the magnitude of the membrane potential and changes in the pH gradient have no significant effect. It is concluded that $\text{Δ}\text{μ}{}_{\mathrm{H}}\text{+}$, more specifically the membrane potential, plays a critical role in Ca⁺⁺-induced transformation.     

The effect of formate on cytochrome aa3 and on electron transport in the intact respiratory chain

1. Formate inhibits cytochrome $\text{c}$ oxidase activity both in intact mitochondria and submitochondrial particles, and in isolated cytochrome $\text{aa}{}_3$. The inhibition increases with decreasing pH, indicating that HCOOH may be the inhibitory species.2. Formate induces a blue shift in the absorption spectrum of oxidized cytochrome $\text{aa}{}_3\text{(a}{}^{\mathrm{3+}}\text{a}{}_3{}^{\mathrm{3+}}$) and in the half-reduced species ($\text{a}{}^{\mathrm{2+}}\text{a}{}_3{}^{\mathrm{3+}}$). Comparison with cyanide-induced spectral shifts, towards the red, indicates that formate and cyanide have opposite effects on the $\text{aa}{}_3$ spectrum, both in the fully oxidized and the half-reduced states. The formate spectra provide a new method of obtaining the difference spectrum of $\text{a}{}_3{}^{\mathrm{2+}}$ minus $\text{a}{}_3{}^{\mathrm{3+}}$, free of the difficulties with cyanide (which induces marked high → low spin spectral shifts in cytochrome $\text{a}{}_3{}^{\mathrm{3+}}$) and azide (which induces peak shifts of cytochrome $\text{a}{}^{\mathrm{2+}}$ towards the blue in both α- and Soret regions).3. The rate of formate dissociation from cytochrome $\text{a}{}^{\mathrm{2+}}\text{a}{}_3{}^{\mathrm{3+}}\text{-}\text{HCOOH}$ is faster than its rate of dissociation from $\text{a}{}^{\mathrm{3+}}\text{a}{}_3{}^{\mathrm{3+}}\text{-}\text{HCOOH}$, especially in the presence of cytochrome $\text{c}$. The $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ for formate inhibition of respiration is a function of the reduction state of the system, varying from 30 mM (100% reduction) to 1 mM (100% oxidation) at pH 7.4, 30 °C.4. Succinate-cytochrome $\text{c}$ reductase activity is also inhibited by formate, in a reaction competitive with succinate and dependent on [formate]².5. Formate inhibition of ascorbate plus $\text{N,N,N′,N′-}\text{tetramethyl}\text{-p-}\text{phenyl-enediamine}$ oxidation by intact rat liver mitochondria is partially released by uncoupler addition. Formate is permeable through the inner mitochondrial membrane and no differences in ‘on’ or ‘off’ inhibition rates were observed when intact mitochondria were compared with submitochondrial particles.6. NADH-cytochrome $\text{c}$ reductase activity is unaffected by formate in submitochondrial particles, but mitochondrial oxidation of glutamate plus malate is subject both to terminal inhibition at the cytochrome $\text{aa}{}_3$ level and to a slow extra inhibition by formate following uncoupler addition, indicating a third site of formate action in the intact mitochondrion.     

Regulation of diamine oxidase expression by β2-adrenoceptors in normal and hypertrophic rat kidney

The administration of preferential adrenergic receptor antagonists to uninephrectomized rats revealed the $\text{β}{}_2\text{-}\text{adrenergic}$ mediation in diamine oxidase activity increase that occurs in the remaining kidney undergoing compensatory hypertrophy. In fact, $\text{β}{}_1\text{,β}{}_2\text{-}$ or $\text{β}{}_2\text{-}$, but not $\text{α}{}_1\text{-}$, $\text{α}{}_2\text{-}$, or $\text{β}{}_1\text{-}\text{receptor-blocking}$ this enzyme enhancement. Further studies with adrenoceptor agonists, such as epinephrine ($\text{α}{}_1$, $\text{α}{}_2$, $\text{β}{}_1$, $\text{β}{}_2$), isoproterenol ($\text{β}{}_1$, $\text{β}{}_2$) or terbutaline ($\text{β}{}_2$) showed that also in normal rat kidney diamine oxidase activity is under the control of $\text{catecholamine}\text{-β}{}_2\text{-}\text{receptors}$ through a mechanism that involves new synthesis of mRNA and protein. Theophylline, an inhibitor of phosphodiesterase, or forskolin, an activator of adenyl cyclase, increased diamine oxidase activity as does epinephrine or nephrectomy. Thus, catecholamine-triggered $\text{β}{}_2\text{-}\text{receptors}$ coupled to adenyl cyclase are involved in the regulation of diamine oxidase activity in normal and hypertrophic rat kidney.     

Effect of para-chloromercuribenzenesulfonic acid and temperature on cell water osmotic permeability of proximal straight tubules

The apparent Arrhenius energy of activation ($\text{E}{}_{\mathrm{<mtext>a</mtext>}}$) of the water osmotic permeability ($\text{P}{}_{\mathrm{<mtext>os</mtext>}}{}^{\mathrm{<mtext>c</mtext>}}$) of the basolateral plasma cell membrane of isolated rabbit proximal straight tubules has been measured under control conditions and after addition of 2.5 mM of the sulfhydryl reagent, para-chloromercuribenzenesulfonic acid (pCMBS), of mersalyl and of dithiothreitol. $\text{E}{}_{\mathrm{<mtext>a</mtext>}}$ (kcal/mol) was $\text{3.2 ± 1.4}$ (controls) and $\text{9.2 ± 2.2}$ (pCMBS), while $\text{P}{}_{\mathrm{<mtext>os</mtext>}}{}^{\mathrm{<mtext>c</mtext>}}$ decreased with pCMBS to $\text{0.26 ± 0.17}$ of its control value. Mersalyl also decreased $\text{P}{}_{\mathrm{<mtext>os</mtext>}}{}^{\mathrm{<mtext>c</mtext>}}$ both in vitro and in vivo (using therapeutical doses). These actions of pCMBS and mersalyl were quickly reverted with 5 mM dithiothreitol and prevented by 0.1 M thiourea. $\text{E}{}_{\mathrm{<mtext>a</mtext>}}$ for free viscous flow is 4.2 and greater than 10 for non-pore-containing lipid membranes. By analogy with these membranes and with red blood cells, where similar effects of pCMBS on $\text{P}{}_{\mathrm{<mtext>os</mtext>}}$ are observed, it is concluded that cell membranes of the proximal tubule are pierced by aqueous pores which are reversibly shut by pCMBS. Part of the action of mercurial diuretics can be explained by their action on $\text{P}{}_{\mathrm{<mtext>os</mtext>}}{}^{\mathrm{<mtext>c</mtext>}}$.     

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.     

Lipid structural order parameters (reciprocal of fluidity) in biomembranes derived from steady-state fluorescence polarization measurements

This paper presents an interpretation of fluorescence polarization measurements in lipid membranes which are labelled with the apolar probe 1,6-diphenyl-1,3,5-hexatriene. The steady-state fluorescence anisotropy, $\text{r}{}_{\mathrm{<mtext>S</mtext>}}$, is resolved into a fast decaying or kinetic component, $\text{r}{}_{\mathrm{<mtext>f</mtext>}}$, and an infinitely slow decaying or static component, $\text{r}{}_{\mathrm{\infty }}$. The latter contribution, which predominates in biological membranes, is exclusively determined by the degree of molecular packing (order) in the apolar regions of the membrane; $\text{r}{}_{\mathrm{\infty }}$ is proportional to the square of the lipid order parameter. An empirical relation between $\text{r}{}_{\mathrm{<mtext>S</mtext>}}$ and $\text{r}{}_{\mathrm{\infty }}$ is presented, which is in agreement with a prediction based on a theory of rotational dynamics in liquid crystals. This relation enabled us to estimate a lipid structural order parameter directly from simple steady-state fluorescence polarization measurements in a variety of isolated biological membranes. It is shown that major factors determining the order parameter in biomembranes are the temperature, the cholesterol and sphingomyelin content and (in a few systems) the membrane intrinsic proteins.     

The polymorphic phase behaviour and miscibility properties of synthetic phosphatidylethanolamines

(1) The polymorphic phase behaviour of aqueous dispersions of various synthetic phosphatidylethanolamines, both singly and in mixtures, has been investigated by ³¹P-NMR. (2) $\text{14:0}\text{14:0}$ PE remains in the lamellar phase up to 90°C. $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibits a lamellar to hexagonal (H_II) transition between 60°C and 63°C. For $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE, the lamellar to hexagonal (H_II) transition occurs between 7 and 12°C, whereas for $\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}$ PE, the hexagonal (H_II) phase is the preferred structure above ?15°C. (3) Mixtures of $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibit near-ideal miscibility behaviour. For mixtures of $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{14:0}\text{14:0}$ PE there is evidence of fluid-solid immiscibility at temperatures below the gel-liquid crystalline transition temperature of the $\text{14:0}\text{14:0}$ PE component. Mixtures of $\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibit complex phase behaviour involving limited fluid-solid immiscibility at low temperatures and formation of a phase allowing isotropic motional averaging at higher temperatures. (4) ³¹P-NMR provides a graphic method for investigating the miscibility properties of mixed PE systems.     

Asymmetric or symmetric? Cytosolic modulation of human erythrocyte hexose transfer

(1) The Michaelis-Menten parameters for hexose transfer in erythroctes, erythrocyte ghosts and inside-out vesicles at 20°C were determined using the light scattering method of Sen and Widdas ((1962) J. Physiol. 160, 392–403). (2) The external $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for infinite-cis exit of d-glucose in cells and ghosts is $\text{3.6 ± 0.5}\text{mM}$. (3) Dilution of cellular solute (up to × 90 dilution) by lysing and resealing cells in varying volumes of lysate is without effect on the $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$ for net d-glucose exit. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for net exit, however, falls from $\text{32.4 ± 3.7}\text{mM}$ in intact cells to $\text{12.9 ± 2.3}\text{mM}$ in ghosts. This effect is reversible. (4) Infinite-cis net d-glucose uptake measurements in cells and ghosts reveal the presence of a low $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, high affinity internal site of $\text{5.9 ± 0.8}\text{mM}$. The $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$ for net glucose entry increases from $\text{23.2 ± 3.7}\text{mmol/l per min}$ in intact cells to $\text{55.4 ± 6.3}\text{mmol/l per min}$ in ghosts. (5) The external $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for infinite-cisd-glucose exit in inside-out vesicles is $\text{6.8 ± 2.7}\text{mM}$. The kinetics of zero-transd-glucose exit from inside-out vesicles are changed markedly when cellular solute (obtained by lysis of intact cells) is applied to either surface of inside-out vesicles. When solute is present externally, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for zero-trans exit are decreased by up to 10-fold. When solute is present at the interior of inside-out vesicles, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for zero-trans exit is reduced; $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for exit is unaffected. In the nominal absence of cell solute, transfer is symmetric in inside-out vesicles. The orientation of transporter in the bilayer is unaffected by the vesiculation procedure. (6) External application of cellular solute to ghosts reduces $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for d-glucose exit but is without effect on the external $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for infinite-cis exit. (7) The inhibitory potency of cell lysate on hexose transfer is lost following dialysis indicating that the factors responsible for transfer modulation are low molecular weight species. (8) We consider the hexose transfer in human erythrocytes is intrinsically symmetric and that asymmetry of transfer is conferred by interaction of the system with low molecular weight cytosolic factors.