Biphasic kinetic plots and specific analogs distinguishing and describing amino acid transport sites in S37 ascites tumor cells

Curve-fitting procedures indicated that exo-2-amino-bicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) modified V and K_m for one of two systems serving for histidine transport into the S37 ascites tumor cells. When this system was obliterated by leucine in the medium, BCH had no effect on histidine transport.Curve-fitting procedures similarly suggest N-methyl-α-aminoisobutyric acid affected the K_m and V values for the other histidine-transporting system and that carboxymethylhistidine (His(Cm)) inhibited both transport systems. His(Cm) further inhibited histidine uptake into leucine-inhibited cells. K_m and V values were altered simultaneously in the presence of several inhibitory analogs.Alanine methyl ester markedly inhibited high-concentration histidine uptake, whereas leucine methyl ester markedly inhibited low-concentration histidine uptake.The present results confirm earlier suggestions that our high c system is Christensen's A system and our low c system his L system. We also confirm a very high degree of specificity of N-methyl-α-aminoisobutyric acid for the A or high c system, and of BCH for the L or low c system. We suggest the utility of combining two approaches to the study of transport system properties; use of specific analogs and modification of biphasic plots. We demonstrate that the carboxyl group is not a prerequisite molecular feature for inhibitory interaction with the A or L system.     

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.     

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.     

Opposite modulation by uncoupling and electron transport limitation of the Kmapp of ADP for photophosphorylation

The $\text{K}{}_{\mathrm{m}}{}^{\mathrm{(app)}}$ of ADP for photophosphorylation in lettuce chloroplasts was measured both at various light intensities and in the presence of various uncoupler (nigericin + K⁺) concentrations. Lowering the light intensity results in both, a decrease in the rate of phosphorylation and a several fold $\text{decrease}$ in the $\text{K}{}_{\mathrm{m}}{}^{\mathrm{(app)}}$ of ADP for the reaction. However, when increasing concentrations of the uncoupler nigericin + K⁺ are employed, the rate of photophosphorylation is decreased but a several-fold $\text{increase}$ in the $\text{K}{}_{\mathrm{m}}{}^{\mathrm{(app)}}$ of ADP for the reaction is observed. The results are discussed in terms of the chemiosmotic hypothesis. It is suggested that these effects might indicate the existence of a mechanism controlling the rate of ATP formation which is different than the formation of the electrochemical gradient.     

The presence of two (Na+ + K+)-ATPase inhibitors in equine muscle ATP: Vanadate and a dithioerythritol-dependent inhibitor

A potent inhibitor of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity was purified from Sigma equine muscle ATP by cation- and anion-exchange chromatography. The isolated inhibitor was identified by atomic absorption spectroscopy and proton resonance spectroscopy to be an inorganic vanadate. The isolated vanadate and a solution of V₂O₅ inhibit sarcolemma $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ with an $\text{I}{}_{50}$ of 1 μM in the presence of 1 mM $\text{ethyleneglycol-bis-(β-aminoethylether)}\text{-N,N′-}\text{tetraacetic}$ acid (EGTA), 145 mM NaCl, 6mM MgCl₂, 15 mM KCl and 2 mM synthetic ATP. The potency of the isolated vanadate in increased by free Mg²⁺. The inhibition is half maximally reversed by 250 μM epinephrine. Equine muscle ATP was also found to contain a second $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ inhibitor which depends on the sulfhydryl-reducing agent dithioerythritol for inhibition. This unknown inhibitor does not depend on free Mg²⁺ and is half maximally reversed by 2 μM epinephrine. Prolonged storage or freeze-thawing of enzyme preparations decreases the susceptibility of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ to this inhibitor. The adrenergic blocking agents, propranolol and phentolamine, do not block the catecholamine reactivation. The inhibitors in equine muscle ATP also inhibit highly purified $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ from shark rectal gland and eel electroplax. The inhibitors in equine muscle ATP have no effect on the other sarcolemmal ATPases, Mg²⁺-ATPase, Ca²⁺-ATPase and $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$.     

Plasmid vectors for high-efficiency expression controlled by the PL promoter of coliphage lambda

A family of plasmid cloning vectors has been constructed that make use of the leftward promoter ($\text{P}{}_{\mathrm{<mtext>L</mtext>}}$) of phage λ to provide for efficient expression of cloned genes in Escherichia coli. The promoter activity of $\text{P}{}_{\mathrm{<mtext>L</mtext>}}$ is fully repressed at low temperature by a thermolabile repressor product of the $\text{λc}\text{I}\text{1857}$ gene, and can be activated by heat induction. Examples are given (β-lactamse, tryptophan synthetase A) where, under optimal conditions, between 30 and 40% of the total protein synthesis is directed by the cloned gene under $\text{P}{}_{\mathrm{<mtext>L</mtext>}}$ control.     

Microcalorimetric studies of the interactions between cytochromes c and c1 and of their interactions with phospholipids

Thermotropic properties of purified cytochrome $\text{c}{}_1$ and cytochrome $\text{c}$ have been studied by differential scanning calorimetry under various conditions. Both cytochromes exhibit a single endothermodenaturation peak in the differential scanning calorimetric thermogram. Thermodenaturation temperatures are ionic strength, pH, and redox state dependent. The ferrocytochromes are more stable toward thermodenaturation than the ferricytochromes. The enthalpy changes of thermodenaturation of ferro- and ferricytochrome $\text{c}{}_1$ are markedly dependent on the ionic strength of the solution. The effect of the ionic strength of solution on the enthalpy change of thermodenaturation of cytochrome $\text{c}$ is rather insignificant. The formation of a complex between cytochromes $\text{c}$ and $\text{c}{}_1$ at lower ionic strength causes a significant destabilization of the former and a slight stabilization of the latter. The destabilization of cytochrome $\text{c}$ upon mixing with cytochrome $\text{c}{}_1$ was also observed at high ionic strength, under which conditions no stable complex was detected by physical separation. This suggests formation of a transient complex between these two cytochromes. When cytochrome $\text{c}$ was complexed with phospholipids, no change in the thermodenaturation temperature was observed, but a great increase in the enthalpy change of thermodenaturation resulted.     

Electrogenic events in the ubiquinone-cytochrome bc2 oxidoreductase of rhodopseudomonas sphaeroides

The reductant of ferricytochrome c₂ in Rhodopseudomonas sphaeroides is a component, Z, which has an equilibrium oxidation-reduction reaction involving two electrons and two protons with a midpoint potential of 155 mV at pH 7. Under energy coupled conditions, the reduction of ferricytochrome c₂ by ZH₂ is obligatorily coupled to an apparently electrogenic reaction which is monitored by a red shift of the endogeneous carotenoids. Both ferricytochrome c₂ reduction and the associated carotenoid bandshift are similarly affected by the concentrations of ZH₂ and ferricytochrome c₂, pH, temperature the inhibitors diphenylamine and antimycin, and the presence of ubiquinone. The second-order rate constant for ferricytochrome c₂ reduction at pH 7.0 and at 24°C was 2 · 109 M^?1 · s^?1, but this varied with pH, being 5.1 · 10⁸ M^?1 · s^?1 at pH 5.2 and 4.3 · 10⁹ M^?1 · s^?1 at pH 9.3. At pH 7 the reaction had an activation energy of 10.3 kcal/mol.     

Depression of amino acid transport in cultured rat hepatocytes by purified enterotoxin from Clostridiumperfringens

Rat liver parenchymal cells (hepatocytes) were isolated by a collagenase perfusion technique and maintained as monolayers in serum-free medium in collagen-coated culture dishes. Glucagon, in combination with dexamethasone, induced α-aminoisobutyric acid transport in these cells. Addition of purified $\text{Clostridium}\text{perfringens}$ enterotoxin to hepatocytes preinduced by glucagon and dexamethasone rapidly depressed (but did not abolish) α-aminoisobutyric acid transport. The toxin effect was dose dependent: 1000 or 300 ng/ml produced maximal depression whereas 100 or 40 ng/ml were without effect in 120 minutes. The effect was eliminated by pretreating the toxin with heat or specific antisera. The effect of enterotoxin on α-aminoisobutyric acid transport in two cultured rat hepatoma cell lines (H4-II-E-C3 and McA-RH 7777) was also investigated. Only the McA-RH 7777 cells were sensitive to the toxin suggesting that the enterotoxin may interact with specific membrane components of normal rat liver cells which are also present on some (but not all) cancerous rat liver cells.     

Transcellular calcium transport by midgut of the blowfly, Calliphoravicina

Transcellular calcium transport by the internally perfused $\text{Calliphora}$ midgut has been measured by simultaneously monitoring ⁴⁵Ca removal from the perfusing saline (entry to the cells) and its appearance in the bathing saline (exit from the cells). Reduction of the Na⁺ gradient across the basolateral membranes of midgut epithelial cells by removal of bathing Na⁺ or by addition of monensin or ouabain inhibits calcium transport across the basolateral membranes. Calcium entry at the apical membranes is inhibited in parallel. The calmodulin inhibitors, trifluoperazine or calmidazolium, do not directly affect calcium transport nor do they dissociate the parallel changes in calcium entry and exit when calcium exit is inhibited. Experiments with A23187 are consistent with a role for intracellular calcium in regulating calcium entry at the apical membranes. It is suggested that calcium transport out of midgut epithelial cells is largely by Na⁺-Ca²⁺ countertransport, and that entry may be regulated by cytoplasmic calcium so that the calcium influx never exceeds the capacity of the transport mechanisms to pump it out of the cells.     

Decreased in vivo protein and phospholipid methylation after in vivo elevation of brain S-adenosyl-homocysteine

The administration of adenosine together with homocysteine resulted in a dose-related elevation of cerebral S-adenosyl-L-homocysteine without concomitant perturbation of S-adenosyl-L-methionine levels. The adenosine + homocysteine treatment also decreased the incorporation of labile and stable methyl groups into brain proteins. Brain [³H]-phosphatidyl N,N-dimethylethanolamine and [³H]-phosphatidylcholine were also significantly decreased while [³H]-phosphatidyl-N-monomethylethanolamine remained unchanged. The data indicate that elevated brain S-adenosylhomocysteine can markedly and selectively inhibit the $\text{in vivo}$ methylation of brain proteins and phospholipids.     

Stimulation of insulin biosynthesis in isolated mouse islets by L-leucine, 2-aminonorbornane-2-carboxylic acid and α-ketoisocaproic acid

An immune binding technique was used for measuring the effects of certain amino acids on the rate of insulin biosynthesis. [³H]phenylalanine served as the radioactive precursor for insulin synthesized by isolated mouse pancreatic islets. L-Leucine was found to stimulate the insulin biosynthesis and this effect was observed already at a physiologic concentration in contrast to the much higher concentrations needed to stimulate insulin secretion in vitro. Furthermore, it was found that 2-aminonorbornane-2-carboxylic acid and α-ketoisocaproic acid shared with glucose and L-leucine the ability to stimulate insulin biosynthesis. In contrast, L-alanine, L-arginine and D-leucine had no stimulatory effect in the absence of glucose, while in the presence of 5 mM glucose L-arginine decreased and L-alanine increased the incorporation rate of tritiated phenylalanine. The fact that many of those compounds which stimulated insulin biosynthesis have also been shown elsewhere to be metabolized by the B-cells supports the view that the rate of insulin biosynthesis may be substrate dependent.     

Bromoacetylcholine as an affinity label of the acetylcholine receptor from Torpedo californica

Bromoacetyl[methyl-³H]choline is a highly specific label for the reduced acetylcholine binding site on the acetylcholine receptor from $\text{Torpedo californica}$. Only one of two binding sites per receptor monomer is susceptible to labeling. The labeled site is on the α chain of the receptor.     

Actions of a nicotinic agonist,DMPP, on intestinal ion transport invitro

High-dose carbachol (10^?3 M) has previously been shown to cause NaCl absorption in short-circuited rabbit ileum. The mechanism of this effect may be norepinephrine release induced by carbachol activation of presynaptic nicotinic receptors on adrenergic neurons. Norepinephrine then interacts with postsynaptic α-adrenergic receptors on intestinal mucosal cells to stimulate neutral NaCl absorption and inhibit electrogenic bicarbonate secretion. The present paper examines the $\text{in vitro}$ intestinal ion transport effects of DMPP an agent which is more specific than carbachol on nicotinic cholinergic receptors. DMPP (10^?5 M) caused a transient increase followed by prolonged depression of the short-circuit current, increased NaCl absorption and increased tissue conductance. This effect was antagonized by hexamethonium and phentolamine. It is concluded that nicotinic cholinergic agents stimulate norepinephrine release from adrenergic nerves and effect intestinal ion transport just as norepinephrine does.     

Mitochondrial calcium flux in temperature-sensitive mutants of Drosophila

The mechanisms of mitochondrial calcium flux in normal and temperature-sensitive mutants of $\text{Drosophila}$ were investigated. Adult mitochondria from all stocks, when analysed with an oxygen electrode, gave respiration rates which exhibited normal responses to adenosine diphosphate and uncoupling agents but no stimulation by calcium. In contrast, calcium stimulates the respiration rate of normal larval mitochondria particularly those of the second instar. This is not evident in second instar mitochondria from the temperature sensitive mutants. There is a rapid accumulation of mitochondrial calcium during normal larval ontogenesis. The levels in temperature-sensitive second instar mitochondria are higher than those of any of the normal larval stages. Adult mitochondria in all cases contain very low levels of calcium. The amount of calcium taken up by mitochondria of second instar temperature-sensitive mutants is lower than that of normal. This may reflect the higher endogenous levels already present in the former. The implications of these variations in calcium metabolism for the behavioural defects of the temperature sensitive mutants is discussed.     

Glycosylation of rat liver cytochrome b5 on the ribosomal level

The distribution and site of synthesis of cytochrome $\text{b}{}_5$ was studied by antibody precipitation of the enzyme labeled $\text{in}\text{vivo}$. The enzyme is present in rough and smooth microsomes, Golgi and outer mitochondrial membranes. The cytochrome is synthesized only on bound ribosomes, where glucosamine and galactose moieties are also added. The enzyme seems to be devoid of mannose and sialic acid residues.