Temperature-sensitive mutations in Drosophila melanogaster. 8. Temperature-sensitive periods of the lethal and morphological phenotypes of selected combinations of notch-locus mutations

Temperature-shift experiments were performed on five Notch-locus genotypes with temperature-sensitive phenotypes. The results show that temperature-sensitive periods (TSPs) for lethality may occur at any developmental stage: (1) $\text{N}{}^{\mathrm{g11}}\text{N}{}^{\mathrm{g11}}$;Dp^51b7 having a short embryonic TSP for lethality, (2) $\text{Ax}{}^{16172}\text{N}{}^{\mathrm{?40}}$ having a second-instar TSP for lethality, and (3) $\text{N}{}^{\mathrm{?103}}\text{fa}{}^{\mathrm{no}}$ with a long, possibly polyphasic, TSP, beginning in the embryonic stage and ending in the pupal stage. On the other hand, TSPs for adult morphological phenotypes appear to be restricted to the third larval instar: (1) $\text{Ax}{}^{16172}\text{N}{}^{\mathrm{?40}}$ having third-instar TSPs for wing vein gapping and ocellar bristle loss, and (2) $\text{N}{}^{\mathrm{?103}}\text{spl}$ having third-instar TSPs for eye facet disarray, wing notching, bristle number variation, and fusion of tarsal segments. The significance of these results is discussed in terms of the role of the Notch locus in development.     

Transfer of N, N'-diacetylchitobiose from dolichyl diphosphate into a gray matter membrane glycoprotein

Gray matter and white matter membranes catalyze the transfer of label from UDP-N-acetyl-[${}^{14}\text{C}$] glucosamine into N-acetyl[${}^{14}\text{C}$]glucosaminyl-pyrophosphoryl-dolichol, N,N′-diacetyl [${}^{14}\text{C}$]chitobiosyl-pyrophosphoryl-dolichol, and N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein. Gel filtration of the Pronase digests of gray matter N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein reveals two N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide fractions. One fraction (A) contains approximately eight glycose units. All of the radioactivity is at nonreducing termini and can be released by treatment with an exo-β-N-acetylglucosaminidase. A smaller N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide (B) is recovered in the elution volume expected for an asparaginyl disaccharide. Structural studies show that the labeled saccharide unit in glycopeptide B is N,N′-diacetyl[${}^{14}\text{C}$]chitobiose. The linkage between the ${}^{14}\text{C}$-labeled disaccharide and the polypeptide has the properties of an N-glycosidic attachment to asparagine. Only the larger N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide (A) is found in Pronase digests of white matter membrane N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein after incubation with UDP-N-acetyl[${}^{14}\text{C}$]glucosamine. When gray matter membranes are incubated with UDP-N-acetyl[${}^{14}\text{C}$]glucosamine in the presence of tunicamycin or UMP, the labeling of glycolipid and the asparaginyl disaccharide is inhibited. UMP and tunicamycin have no effect on the transfer of N-acetyl[${}^{14}\text{C}$]glucosamine to external acceptor sites of the larger glycopeptide (A). The transfer of N,N′-diacetyl[${}^{14}\text{C}$]-chitobiose from carrier lipid to protein is observed when extensively washed membranes containing endogenous, prelabeled ${}^{14}\text{C}$-labeled glycolipids are incubated in the presence or absence of unlabeled GDP-mannose. UMP treatment of the prelabeled membranes selectively discharged over 80% of the label from N-acetyl[${}^{14}\text{C}$]glucosaminyl-pyrophosphoryl-dolichol, but had no effect on the transfer of the ${}^{14}\text{C}$-labeled disaccharide to protein. All of these results are concordant with transfer of N,N′-diacetylchitobiose from dolichyl diphosphate to gray matter glycoprotein. The major membrane glycoprotein labeled by the lipid-mediated [${}^{14}\text{C}$]disaccharide transfer reaction has an apparent molecular weight of 24,000. Tunicamycin prevents the enzymatic labeling of the gray matter glycoprotein having an apparent molecular weight of 24,000.     

Properties of bilayer membranes in the presence of dipicrylamine. A comparative study by optical absorption and electrical relaxation measurements

In order to test the question if a pool of lipophilic ions may exist in black lipid membranes which cannot be detected by electrical relaxation measurements we have performed simultaneously measurements of the optical absorption of a lipophilic ion. The absorbance of membrane-bound dipicrylamine at 410 nm was measured with a sensitive spectrophotometer which can detect absorbance changes $\text{? 4 · 10}{}^{\mathrm{?5}}$. A minimal concentration of about 6 · 10¹¹ dipicrylamine ions per cm² of the membrane could be detected with this instrument. The dipicrylamine concentration in the membrane obtained with the optical method $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>opt</mtext>}}$ is compared with the concentrations $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>el</mtext>}}$ obtained from simultaneous electrical relaxation measurements. $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>opt</mtext>}}$ and $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>el</mtext>}}$ agreed at low dipicrylamine concentrations (10^?8–10^?7 M in the aqueous phase) and showed saturation at higher concentrations (up to 5 · 10^?6 M). In the saturation range $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>opt</mtext>}}$ was maximally four times higher than $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>el</mtext>}}$. The significance of this difference is discussed together with general aspects of the saturation phenomenon.     

Chemical and spectroscopic conformation of an exogenous ligand bridge in half met hemocyanin.

Half $\text{met-N}{}_3{}^{\mathrm{?}}$ hemocyanin is shown to undergo a unique change at the Cu(II)?Cu(I) active site with temperature, exhibiting class II mixed valent properties at low temperature (The appearance of an intense near IR intervalence-transfer transition and a delocalized EPR spectrum). This requires a Cu(II)NNNCu(I) bridging geometry. The effects of CO coordination to half $\text{met-N}{}_3{}^{\mathrm{?}}$, combined with the presence of a low energy $\text{N}{}_3{}^{\mathrm{?}}\text{→ Cu(II)}$ charge transfer transition, demonstrate that azide is also bridging at room temperature. Finally, half $\text{met-N}{}_3{}^{\mathrm{?}}$ is found to be capable of coordination of a second $\text{N}{}_3{}^{\mathrm{?}}$ at the copper(II) site.     

Picosecond laser study of fluorescence lifetimes in spinach chloroplast Photosystem I and Photosystem II preparations

Fractions enriched in either Photosystem I or Photosystem II have been prepared from chloroplasts with digitonin. A more detailed analysis of the decay kinetics of fluorescence excited by a picosecond laser pulse has been possible compared to experiments with unfractionated systems. The Photosystem I fractions show a very short component (? 100 ps) at room temperature which is apparently independent of pulse intensity over the range of photon densities used (5 · 10¹³–1 · 10¹⁶ photons cm^?2). The Photosystem II fraction has a short initial lifetime at room temperature which is strongly intensity-dependent approaching 500 ps at low photon densities, but decreasing to close to 150 ps at the highest photon densities. All of these room temperature decays appear to be non-exponential, and may possibly be fitted by at $\text{t}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ expression, expected from a random diffusion of excitations via Förster energy transfer. On cooling to 77 K, lifetimes of both Photosystem I and Photosystem II increase, the lengthening with Photosystem I being more striking. The Photosystem I decays become intensity dependent like the Photosystem II, and at the lowest photon densities decays which are more nearly exponential within the experimental error give initial lifetimes of about 2 ns. The non-exponential decays seen at high photon densities appear to fit a $\text{t}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ expression.     

A high-affinity (Ca2+ + Mg2+)-ATPase in plasma membranes of rat ascites hepatoma AH109A cells

The activity of calcium-stimulated and magnesium-dependent adenosinetriphosphatase which possesses a high affinity for free calcium (high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$, EC 3.6.1.3) has been detected in rat ascites hepatoma AH109A cell plasma membranes. The high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ had an apparent half saturation constant of $\text{77 ± 31}\text{nM}$ for free calcium, a maximum reaction velocity of $\text{9.9 ± 3.5}\text{nmol}$ ATP hydrolyzed/mg protein per min, and a Hill number of 0.8. Maximum activity was obtained at 0.2 μM free calcium. The high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ was absolutely dependent on 3–10 mM magnesium and the pH optimum was within physiological range (pH 7.2–7.5). Among the nucleoside trisphosphates tested, ATP was the best substrate, with an apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 30 μM. The distribution pattern of this enzyme in the subcellular fractions of the ascites hepatoma cell homogenate (as shown by the linear sucrose density gradient ultracentrifugation method) was similar to that of the known plasma membrane marker enzyme alkaline phosphatase (EC 3.1.3.1), indicating that the ATPase was located in the plasma membrane. Various agents, such as K⁺, Na⁺, ouabain, KCN, dicyclohexylcarbodiimide and NaN₃, had no significant effect on the activity of high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$. Orthovanadate inhibited this enzyme activity with an apparent half-maximal inhibition constant of 40 μM. The high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ was neither inhibited by trifluoperazine, a calmodulin-antagonist, nor stimulated by bovine brain calmodulin, whether the plasma membranes were prepared with or without ethylene glycol $\text{bis}\text{(β-}\text{aminoethyl ether}\text{)-N,N,N′,N′-}\text{tetraacetic acid}$. Since the kinetic properties of the high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ showed a close resemblance to those of erythrocyte plasma membrane $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$, the high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ of rat ascites hepatoma cell plasma membrane is proposed to be a calcium-pumping ATPase of these cells.     

Magnetic resonance study of 13C reductively methylated glycophorin and glycophorin glycopeptides

¹³C nuclear magnetic resonance (n.m.r.) spectral data for ¹³C reductively methylated N-terminal tryptic glycopeptides and for ¹³C reductively methylated N-terminal glyco-octapeptides derived from homozygous glycophorins A^M and A^N are presented. Their ¹³C chemical shift data are compared with the previously published ¹³C n.m.r. data for ¹³C reductively methylated homozygous glycophorins A^M and A^N in order to investigate the means of display of the MN blood determinants by these species. The pH dependence of the ¹³C resonances of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ leucine of glyco-octapeptide A^N and of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ serine of glyco-octapepti A^M indicated that only a slight structural perturbation occurs at the N-terminus when a large portion of the glycoprotein molecule is removed. However, one structural ‘state’ of ¹³C reductively methylated glycophorin A^M is lost when the glyco-octapeptide A^M is produced. The ¹³C resonance of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ leucine of glycooctapeptide A^N titrated with a $\text{p}\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ of 7.7 (Hill coefficient $\text{～ 1}$). The ¹³C resonance of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ serine, on the other hand, exhibited an unusual pH dependence, indicating the existence of some possible steric constraints or hydrogen bonding in this molecule. In comparison to the data obtained for ¹³C-labelled glycooctapeptide A^M molecule, the pH dependence of the chemical shift of the ¹³C resonance of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ serine of tripeptide tri-L-serine is also presented. Circular dichroism (c.d.) spectra indicated that the reductive methylation technique does not cause a large perturbation of the glycophorin A molecule.     

Respiration-driven proton translocation with nitrite and nitrous oxide in Paracoccus denitrificans

(1) $\text{H}$⁺/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O₂, NO^?₂ or N₂O. Under optimal H⁺-translocation conditions, the ratios $\text{H}{}^{\mathrm{+}}\text{O}$, $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as exogenous substrate, addition of NO^?₂ or N₂O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were ?0.84, ?2.33 and ?1.90, respectively. (3) The $\text{H}{}^{\mathrm{+}}\text{oxidant}$ ratios, mentioned in item 2, were not altered in the presence of $\text{valinomycin}\text{K}{}^{\mathrm{+}}$ and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O₂, NO^?₂ and N₂O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO^?₂, N₂O or O₂ pass two H⁺-translocating sites. The $\text{H}{}^{\mathrm{+}}\text{electron}$ acceptor ratios predicted by this scheme are in good agreement with the experimental values.     

The effects of membrane lipid order and cholesterol on the internal and external cationic sites of the Na+−K+ pump in erythrocytes

Cholesterol depletion alters the apparent affinity of the internal cationic sites and the maximal translocation rate but not the affinity of the external cationic sites of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump in human erythrocytes. To test whether these effects were mediated by a direct cholesterol-internal site interaction or by a change in membrane lipid order, the effects of five fluidizing amphiphiles (chlorpromazine, imipramine, benzyl alcohol, sodium oleate and sodium benzenesulphonate) on the kinetic parameters of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump were determined. The cholesterol removal and all the agents used induced dose-response decreases in membrane lipid order as measured by fluorescence polarization or ESR. Positive and neutral amphiphiles mimicked the effects of cholesterol removal on the affinity of the internal sites of the pump and to a lesser extent on the maximal translocation rate. Anionic amphiphiles had no effect on internal sites, probably because they distributed preferentially within the outer leaflet on the membrane. These results indicate that cholesterol controls the affinity of the internal sites of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump by altering the membrane lipid order. In contrast, neither cholesterol depletion nor the agents used altered the affinity of the external sites of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump. This difference in sensitivity to membrane lipid order suggests that internal and external cationic sites, although borne by the same protein, are in different lipid environments.     

The insect brain (Na+ + K+)-ATPase Binding of ouabain in the hawk moth, Manduca sexta

(1) A quantitative study has been made of the binding of ouabain to the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ will bind ouabain either in the presence of Mg²⁺ and P_i, (‘Mg²⁺, P_i’ conditions) or in the presence of Na⁺, Mg²⁺, and an adenine nucleotide (‘nucleotide’ conditions) as is the case for the bovine brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the $\text{K}{}_{\mathrm{<mtext>D</mtext>}}$) of ouabain to the M. sexta brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ under both binding conditions. However, ouabain binding is more sensitive to K⁺ inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K⁺ inhibition under the both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ has a higher $\text{K}{}_{\mathrm{<mtext>D</mtext>}}$ for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ can bind ouabain.     

Oxidative phosphorylation by membrane vesicles from Bacillus alcalophilus

ADP and P_i-loaded membrane vesicles from l-malate-grown Bacillus alcalophilus synthesized ATP upon energization with $\text{ascorbate}\text{N,N,N′,N′-}\text{tetramethyl}\text{-p-}\text{phenylenediamine}$. ATP synthesis occurred over a range of external pH from 6.0 to 11.0, under conditions in which the total protonmotive force was as low as ?30 mV. The phosphate potentials (ΔG_p) were calculated to be 11 and 12 kcal/mol at pH 10.5 and 9.0, respectively, whereas the values in vesicles at these two pH values were quite different (?40 ± 20 mV at pH 10.5 and ?125 ± 20 mV at pH 9.0). ATP synthesis was inhibited by KCN, gramicidin, and by N,N′-dicyclohexylcarbodiimide. Inward translocation of protons, concomitant with ATP synthesis, was demonstrated using direct pH monitoring and fluorescence methods. No dependence upon the presence of Na⁺ or K⁺ was found. Thus, ATP synthesis in B. alcalophilus appears to involve a proton-translocating ATPase which functions at low .     

Purification from brain of an intrinsic membrane protein fraction enriched in (Na+ + K+)-ATPase

A microsomal fraction from canine brain gray matter has been extracted with the detergent sodium dodecyl sulfate to partially purify the membrane bound $\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-stimulated}$ adenosine triphosphatase. Phospholipid, glycolipid, and a family of other glycoproteins are also enriched by the procedure; it is proposed that the product is an intrinsic membrane protein fraction. 6–8-fold purification of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ is obtained without solubilizing the enzyme and without irreversibly altering its turnover number. Final specific activities are 350–400 μmol of ATP hydrolyzed/h per mg protein. The stimulation and reversible inactivation of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ by dodecyl sulfate were examined for information relevant to the mechanism of action of the detergent.     

Saturation transfer electron paramagnetic resonance on membrane bound proteins. II-Absence of rotational diffusion of the cholinergic receptor protein in Torpedo marmorata membrane fragments.

We have applied the technique of saturation transfer electron paramagnetic resonance to study the rotational diffusion of spin labeled membrane bound cholinergic receptors from Torpedo marmorata. Two different spin labels were used: a spin labeled maleimide derivative which binds covalently to proteins and a long chain spin labeled acylcholine which binds reversibly with a high affinity to the receptor protein. The maleimide spin label has a motion whose rotational correlation time is τ₂ > 10^?3 sec. The long chain spin labeled acylcholine indicates slightly more motion ($\text{τ}{}_2\text{? 10}{}^{\mathrm{?4}}\text{sec}$), but the nitroxide in this latter case is probably more loosely bound.     

Modification of the aggregation state of the multifunctional enzyme complex catalyzing the first steps in pyrimidine biosynthesis in the course of development of Drosophila melanogaster

Mutations at the bithoraxoid (bxd) and postbithorax (pbx) loci cause a transformation of posterior haltere to posterior wing. It has previously been shown that pbx and $\text{pbx}\text{Ubx}{}^{101}$ cause this transformation by affecting the maintenance (or cell heredity function) of determination so that the transformed cells are indistinguishable from normal wing cells, and have no “memory” of having been part of a haltere disk (Adler, 1978a). I report here that Tp(3) $\text{bxd}{}^{100}\text{Ubx}{}^{101}$ and bxd¹, pbx, e^w both cause the transformation of posterior haltere to posterior wing in the same way as pbx. On the other hand, bxd¹, $\text{bxd}{}^1\text{Ubx}{}^{101}$, bxd^51j, $\text{bxd}{}^{\mathrm{<mtext>51</mtext><mtext>j</mtext>}}\text{Ubx}{}^{101}$, and $\text{bxd}{}^{\mathrm{<mtext>51</mtext><mtext>j</mtext>}}\text{red pbx}$ cause this same transformation of posterior haltere to posterior wing by interfering with the expression of the determined state so that the developmental information of posterior haltere is “misread” as posterior wing. The transformed cells in these disks retain the memory of having been part of a haltere disk; that is, these posterior cells that would secrete wing cuticle during metamorphosis regenerate anterior haltere structures. Thus it appears clear that it is possible to uncouple the expression and cell heredity functions of determination in the haltere disk of Drosophila.     

Harmaline distribution in single muscle fibres and the inhibition of sodium efflux

Harmaline, a known inhibitor of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in cell membranes, inhibited 50% of the ²²Na efflux from barnacle muscle fibres at an extracellular concentration of 2.4 mM. Injected harmaline inhibited 50% of the efflux at an estimated intracellular concentration of about $\text{8}\text{mM}\text{·}\text{kg}{}^{\mathrm{?1}}$, assuming complete equilibration with no binding. Total fibre harmaline was measured in separate fibres by ultraviolet spectrophotometry. Fibres in 3 mM harmaline saline accumulated harmaline with a half-time of 17 min and a final total fibre concentration of $\text{6–12}\text{mM}\text{·}\text{kg}{}^{\mathrm{?1}}$. In harmaline-free saline this accumulated harmaline was lost exponentially with a half-time of 35 min; injected harmaline was lost exponentially from fibres with a half-time of 50 min. It is proposed that harmaline crosses the fibre membrane as the uncharged base and that its apparent accumulation against a concentration gradient is mainly due to intracellular binding with an additional contribution from a transmembrane pH gradient. It is concluded that, in fibres exposed to harmaline saline, the intracellular concentration can reach a sufficiently high value, as judged from the results of the injection experiments, to inhibit Na⁺ efflux at an interior-facing site on the fibre membrane. In contrast, harmaline appears to inhibit the Na⁺-dependent uptake of l-glutamate at an extracellular site.     

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.     

The surface membrane of the small intestinal epithelial cell. I. Localization of adenyl cyclase

The subcellular distribution of adenyl cyclase was investigated in small intestinal epithelial cells. Enterocytes were isolated, disrupted and the resulting membranes fractionated by differential and sucrose gradient centrifugation. Separation of luminal (brush border) and contra-luminal (basolateral) plasma membrane was achieved on a discontinuous sucrose gradient.The activity of adenyl cyclase was followed during fractionation in relation to other enzymes, notably those considered as markers for luminal and contraluminal plasma membrane. The luminal membrane was identified by the membrane-bound enzymes sucrase and alkaline phosphatase and the basolateral region by ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase. Enrichment of the former two enzymes in purified luminal plasma membrane was 8-fold over cells and that of ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase in purified basolateral plasma membranes was 13-fold. F^?-activated adenyl cyclase co-purified with ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase, suggesting a common localization on the plasma membrane. The distribution of K⁺-stimulated phosphatase and 5′-nucleotidase also followed ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase during fractionation.     

Letter: Conformation of thyrotropin-releasing hormone: prediction from molecular orbital theory

The extended Huckel molecular orbital method was used to predict the preferred conformation of thyrotropin releasing hormone (TRH). Calculation of the whole molecule confirmed the conformational preferences of the individual amino acid residues calculated separately. A stereochemical basis is predicted for the differing activity of $\text{N}{}^1{}^{\mathrm{im}}$ methyl and $\text{N}{}^3{}^{\mathrm{im}}$ methyl TRH analogues.