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 $\text{P}{}_{\mathrm{<mtext>os</mtext>}}{}^{\mathrm{<mtext>c</mtext>}}$.     

Characterization of cytochrome P-450SCC-containing liposomes

Purified cytochrome $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ from bovine adrenocortical mitochondria was incorporated into liposomes by the cholate-dilution method utilizing either dialysis or Sephadex gel filtration. Among synthetic phospholipids tested, dioleoylglycerophosphocholine showed the best stability during the incorporation of $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ into liposomes. A maximum amount of heme was incorporated into liposomes at a molar ratio of phospholipid to the cytochrome of approx. 200. When $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ was incorporated into the dioleoylglycerophosphocholine liposomes by the cholate-filtration method, the $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}\text{-}\text{containing liposomes}$ showed two major populations on the elution pattern of the Sepharose 4B gel filtration, and were seen at a diameter of 200–600 Å and its aggregated forms. When the cytochrome was incorporated into dioleoylglycerophosphocholine liposomes or cholesterol-free adrenocortical mitochondrial liposomes, $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ was less stable than $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ in aqueous solution. Cholesterol or adrenodoxin markedly stabilized the liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$. Liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ required cholesterol for its optimum reduction with adrenodoxin, adrenodoxin reductase, and NADPH in the presence of CO. About 70% of the total heme in the dioleoylglycerophosphocholine liposomes was reduced by the enzymatic reduction in the presence of cholesterol, indicating that 70% of the total molecules are exposed to the surface of the outer monolayer. In order to see the location of the heme in membrane, the dioleoylglycerophosphocholine-liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ was subjected to $\text{p-}\text{chloromercuriphenyl sulfonic acid}$ treatment. This reagent destroyed the liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$. These results suggest that the heme is located in the proximity of the $\text{p-}\text{chloromercuriphenyl sulfonic acid}$ reacting sites which are exposed to the surface, or located on the vincinity of polar heads of the membrane.     

Optimizing enzyme assays with one or two coupling enzymes

The cost of assays using one or two coupling enzymes is optimized by using equations to calculate the minimum amount(s) of enzyme(s) which should be used to obtain a given time (t₉₉) in which 99% of the rate V₀ of the first reaction is obtained. Using two coupling enzymes and given a value of t₉₉, the induction period L = L₁ + L₂ fulfills the requirement $\text{t}{}_{99}\text{2}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{4.6}\text{≥ L ≥}\text{t}{}_{99}\text{4.6}$, allowing one to choose a cost lower than that derived from the until-now generally applied assumption of t₉₉ = 4.6L. Being $\text{α =}\text{L}{}_1\text{L}{}_2$, in optimized assays the values α, t₉₉, and L are related by $\text{T}{}_{99}\text{=4.6}\text{(1+α)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{1+α}\text{L}$, thus allowing (graphical) calculation of the amounts of coupling enzymes which will minimize the cost for every chosen t₉₉ or L. Maximum practical rates, allowed in some supposed interesting cases, have been calculated.     

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.     

Correlation of redox levels of component electron carriers with total electron flux in an electron-transport system. P-700 and the photoreduction of NADP+ in chloroplast fragments

A mathematical analysis is described which measures the effects of actinic light intensity and concentration of an artificial electron donor on the steady-state light-induced redox level of a reaction-center pigment (e.g. P-700) and on the overall light-induced electron flux (e.g. reduction of NADP⁺). The analysis led to a formulation (somewhat similar to the Michaelis-Menten equation for enzyme kinetics) in which a parameter, $\text{I}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, is defined as the actinic light intensity that, at a given concentration of electron donor, renders the reaction-center pigment half oxidized and half reduced. To determine the role of a presumed reaction-center pigment, $\text{I}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ is compared with another parameter, equivalent to $\text{I}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, that is obtained independently of the reaction-center pigment by measuring the effect of actinic light intensity and concentration of electron donor on the overall electron flow.The theory was tested and validated in a model system with spinach Photosystem I chloroplast fragments by measurements of photooxidation of P-700 and light-induced reduction of NADP⁺ by reduced 2,6-dichlorophenolindophenol. A possible extension of this mathematical analysis to more general electron-transport systems is discussed.     

Studies of the nucleotide-binding sites on the mitochondrial F1-ATPase through the use of a photoactivable derivative of adenylyl imidodiphosphate

(1) $\text{N-4-}\text{Azido}\text{-2-}\text{nitrophenyl}\text{-γ-[}{}^3\text{H]aminobutyryl-Ado}\text{PP[}\text{NH}\text{]P(}\text{NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P)}$ a photoactivable derivative of 5-adenylyl imidodiphosphate (AdoPP[NH]P), was synthesized. (2) Binding of ³H]NAP₄-AdoPP[NH]P to soluble ATPase from beef heart mitochrondria (F₁) was studied in the absence of photoirradiation, and compared to that of $\text{[}{}^3\text{H]Ado}\text{PP[}\text{NH}\text{]P}$. The photoactivable derivative of AdoPP[NH]P was found to bind to F₁ with high affinity, like AdoPP[NH]P. Once $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ had bound to F₁ in the dark, it could be released by AdoPP[NH]P, ADP and ATP, but not at all by NAP₄ or AMP. Furthermore, preincubation of F₁ with unlabeled AdoPP[NH]P, ADP, or ATP prevented the covalent labeling of the enzyme by $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ upon photoirradiation. (3) Photoirradiation of F₁ by $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ resulted in covalent photolabeling and concomitant inactivation of the enzyme. Full inactivation corresponded to the binding of about 2 mol $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}\text{mol F}{}_1$. Photolabeling by NAP₄-AdoPP[NH]P was much more efficient in the presence than in the absence of MgCl₂. (4) Bound $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ was localized on the α- and β-subunits of F₁. At low concentrations (less than 10 μM), bound $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ was predominantly localized on the α-subunit; at concentrations equal to, or greater than 75 μM, both α- and β-subunits were equally labeled. (5) The extent of inactivation was independent of the nature of the photolabeled subunit (α or β), suggesting that each of the two subunits, α and β, is required for the activity of F₁. (6) The covalently photolabeled F₁ was able to form a complex with aurovertin, as does native F₁. The ADP-induced fluorescence enhancement was more severely inhibited than the fluorescence quenching caused by ATP. The percentage of inactivation of F₁ was virtually the same as the percentage of inhibition of the ATP-induced fluorescence quenching, suggesting that fluorescence quenching is related to the binding of ATP to the catalytic site of F₁.     

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

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

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 .     

A convective mass transfer model for determining intestinal wall permeabilities: laminar flow in a circular tube

A convective mass transfer model as analyzed and developed for use in determining intestinal wall permeabilities from external perfusion experiments. Analysis of the model indicates that the ratio of the exit to inlet concentration $\text{C}{}_{\mathrm{m}}\text{C}{}_0$ is a function of only two dimensionless independent variables, the wall permeability, $\text{P}{}_{\mathrm{w}}{}^1$ and Graetz number, Gz = πDL/2Q. The Graetz number contains the independent variables of interest, length, diflusivity, and flow rate. The radius of the intestine is included implicitly in the flow rate. Since $\text{C}{}_{\mathrm{m}}\text{C}{}_0$ and Gz are the experimental quantities, and the solution to the model system contains P_ω¹ implicitly, a convenient approximate method is developed which allows a direct calculation of P_ω¹. This method is in error by 10–20% in the worst cases. The approach is illustrated by application to the determination of the wall permeabilities for two non polar compounds.     

Sucrose transport by Streptococcus mutans. Evidence for multiple transport systems

The transport of sucrose by selected mutant and wild-type cells of Streptococcus mutans was studied using washed cocci harvested at appropriate phases of growth, incubated in the presence of fluoride and appropriately labelled substrates. The rapid sucrose uptake observed cannot be ascribed to possible extracellular formation of hexoses from sucrose and their subsequent transport, formation of intracellular glycogen-like polysaccharide, or binding of sucrose or extracellular glucans to the cocci. Rather, there are at least three discrete transport systems for sucrose, two of which are $\text{phospho}\text{enol}\text{pyruvate-dependent}$ phosphotransferases with relatively low apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values and the other a non-phosphotransferase (non-PTS) third transport system (termed TTS) with a relatively high apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. For strain 6715-13 mutant 33, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values are 6.25·10^?5 M, 2.4·10^?4 M, and 3.0·10^?3 M, respectively; for strain NCTC-10449, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values are 7.1·10^?5 M, 2.5·10^?4 M and 3.3·10^?3 M, respectively. The two lower $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ systems could not be demonstrated in mid-log phase glucose-adapted cocci, a condition known to repress sucrose-specific phosphotransferase activity, but under these conditions the highest $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ system persists. Also, a mutant devoid of sucrose-specific phosphotransferase activity fails to evidence the two high affinity (low apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$) systems, but still has the lowest affinity (highest $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$) system. There was essentially no uptake at 4°C indicating these processes are energy dependent. The third transport system, whose nature is unknown, appears to function under conditions of sucrose abundance and rapid growth which are known to repress $\text{phospho}\text{enol}\text{pyruvate-dependent}$ sucrose-specific phosphotransferase activity in S. mutans. These multiple transport systems seem well-adapted to S. mutans which is faced with fluctuating supplies of sucrose in its natural habitat on the surfaces of teeth.     

The covariance of relatives derived from a random mating population.

Attention is drawn to errors common in the derivation of forms for the genotypic covariance of noninbred relatives from a Hardy-Weinberg population of diploids. A synthesis of Fisher's least-squares method of partitioning the genotypic variance and Malécot's probability method of expressing kinship, yields a general form. For one locus, the form is $\text{(P}{}_{\mathrm{ss}}\text{+ P}{}_{\mathrm{sd}}\text{+ P}{}_{\mathrm{ds}}\text{+ P}{}_{\mathrm{dd}}\text{)}\text{1}\text{2}\text{σ}{}_{\mathrm{a}}{}^2\text{+ (P}{}_{\mathrm{ss}}\text{P}{}_{\mathrm{dd}}\text{+ P}{}_{\mathrm{sd}}\text{P}{}_{\mathrm{ds}}\text{) σa}{}_{\mathrm{d}}{}^2$, where σ_a² is the additive genetic variance, α_d² is the variance of dominance deviations, p_ij is the probability that parental gamete i is identical by descent to parental gamete j, i = s, d indexes the parents of one relative, and j = s, d indexes those of the other. The form provides a framework for obtaining the covariance of relatives from an equilibrium population with linkage.     

Study on models of single populations: an expansion of the logistic and exponential equations

Using the adsorption theory of chemical kinetics, a new equation concerning the growth of single populations is presented:

$\text{d}\text{X}\text{d}\text{t}\text{=}\text{μ}{}_{\mathrm{c}}\text{X(1 ?)}\text{X}\text{X}{}_{\mathrm{m}}\text{1?}\text{X}\text{X}{}_{\mathrm{m}}{}^{\mathrm{\prime }}$

or in its integral form:

$\text{ln}\text{X}\text{X}{}_{\mathrm{o}}\text{?}\text{ln}\text{X}{}_{\mathrm{m}}\text{?X}\text{X}{}_{\mathrm{m}}\text{?X}{}_{\mathrm{o}}\text{+}\text{X}{}_{\mathrm{m}}\text{X}{}^{\mathrm{\prime }}{}_{\mathrm{m}}\text{X}{}_{\mathrm{m}}\text{?X}\text{X}{}_{\mathrm{m}}\text{?X}{}_{\mathrm{o}}\text{=μ}{}_{\mathrm{c}}\text{(t?t}{}_{\mathrm{o}}\text{)}$

This equation attempts to explain the relationship between population increment and limiting resources. It can be reduced to either the logistic or exponential equation under two extreme conditions. The new equation has three parameters, X_m, X′_m and μ_c, each of which has ecological significance. $\text{X}{}_{\mathrm{m}}\text{X′}{}_{\mathrm{m}}$ concerns the efficiency of nutrient utilization by an organism. Its value is between zero and one. With ratios approaching unity, the efficiency is high; lower ratios indicate that population increment is quickly restricted by limiting resources. μ_c, is a velocity parameter lying between μ_e, (exponential growth) and μ_L (logistic growth), and is dependent on the value of $\text{solX}{}_{\mathrm{m}}\text{X′}{}_{\mathrm{m}}$. From μ_c we can predict the time course of population incremental velocity ($\text{d}\text{X}\text{d}\text{t}$), and can observe that it is not symmetrical, unlike that derived from the logistic equation. At $\text{X}{}_{\mathrm{m}}\text{X′}{}_{\mathrm{m}}\text{= 1}$ the maximum velocity of the population increment predicted from the new equation is twice that of the logistic equation.Population growth in nature seems to support the new equation rather than the logistic equation, and it can be successfully fitted by means of a least square method.     

Gene m3 mediated efflux of intracellular leucine from bacteriophage P22 infected Salmonella typhimurium

Infection of $\text{Salmonella}\text{typhimurium}$ with the $\text{m}{}_3$ mutant of bacteriophage P22 leads to a rapid and severe efflux of intracellular leucine. The superinfection exclusion ($\text{sie}$) genes of P22 interfere with the function of $\text{m}{}_3$ gene, the product(s) of which is speculated to be an internal protein of phage P22.     

Ammonium (methylammonium) transport by Klebsiella pneumoniae

Klebsiella pneumoniae can accumulate methylammonium up to 80-fold by means of a transport system as indicated by the energy requirement, saturation kinetics and a narrow pH profile around pH 6.8. Methylammonium transport (apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 100 μ}\text{M}$, $\text{V = 40 μ}\text{mol/min}$ per g dry weight at 15°C) is competitively inhibited by ammonium (apparent $\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 7 μ}\text{M}$). The low $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ value and the finding that methylammonium cannot serve as a nitrogen source indicate that ammonium rather than methylammonium is the natural substrate. Uphill transport is driven by a component of the protonmotive force, probably the membrane potential. The transport system is under genetic control; it is partially repressed by amino acids and completely by ammonium. Analysis of mutants suggest that the synthesis of the ammonium transport system is subject to the same ‘nitrogen control’ as nitrogenase and glutamine synthetase.     

Cockroaches on a treadmill: Aerobic running

Five species of cockroach were tested on a miniature treadmill at three velocities as O₂ consumption () was measured: Gromphadorhina chopardi, Blaberus discoidalis, Eublaberus posticus, Byrsotria fumagata and Periplaneta americana. All cockroaches showed a classical aerobic response to running: increased rapidly from a resting rate to a steady-state on-response varied from under 30 s to 3 min. Recovery after exercise was rapid as well; $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ off-response varied from under 30 s to 6 min. These times are faster or similar to mammalian values. varied directly with velocity as in running mammals, birds and reptiles. during steady-state running was only 4–12 times higher than at rest. Running is energetically much less costly per unit time than flying, but the cost of transport per unit distance is much more expensive for pedestrians. The minimal cost of transport (M_run), the lowest necessary to transport a given mass a specific distance, is high in cockroaches due to their small size. The new data suggest that insects may be less economical than comparable sized vertebrates.     

The diffusion-concentration product of oxygen in lipid bilayers using the spin-label T1 method

A method is described to measure the oxygen diffusion-concentration product, $\text{D}{}_{\mathrm{<mtext>o</mtext>}}\text{[}\text{O}{}_2\text{]}$, at any locus that can be probed or labeled using nitroxide radicals. The method is based on the dependence of the spin-lattice relaxation time $\text{T}{}_1$ of the spin label on the bimolecular collision rate with oxygen. Strong Heisenberg exchange between spin label and oxygen contributes directly to $\text{T}{}_1$ of the spin label, while dipolar interactions are negligible. Both time-domain and continuous wave saturation methods for studying $\text{T}{}_1$ are considered. The method has been applied to phospholipid liposomes using fatty acid spin labels. A discontinuity in $\text{D}{}_{\mathrm{<mtext>o</mtext>}}\text{[}\text{O}{}_2\text{]}$ at the main phase transition was observed.     

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.     

Effects of oligomycin and quercetin on the hydrolytic activities of the (Na+ + K+)-dependent ATPase

Quercetin inhibited a dog kidney ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ preparation without affecting $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP or $\text{K}{}_{0.5}$ for cation activators, attributable to the slowly-reversible nature of its inhibition. Dimethyl sulfoxide, a selector of E₂ enzyme conformations, blocked this inhibition, while the K⁺-phosphatase activity was at least as sensitive to quercetin as the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity, all consistent with quercetin favoring E₁ conformations of the enzyme. Oligomycin, a rapidly-reversible inhibitor, decreased the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP and the $\text{K}{}_{0.5}$ for cation activators, and its inhibition was also diminished by dimethyl sulfoxide. Although oligomycin did not inhibit the K⁺-phosphatase activity under standard assay conditions, a reaction presumably catalyzed by E₂ conformations, its effects are nevertheless accommodated by a quantitative model for that reaction depicting oligomycin as favoring E₁ conformations. The model also accounts quantitatively for effects of both dimethyl sulfoxide and oligomycin on $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for substrate, and $\text{K}{}_{0.5}$ for K⁺, as well as for stimulation of phosphatase activity by both these reagents at low K⁺ but high Na⁺ concentrations.