Studies on glutathione transport utilizing inside-out vesicle prepared from human erythrocytes

Adenosine triphosphate-dependent glutathione transport was characterized using inside-out vesicles made from human erythrocytes. Kinetic analysis of the glutathione disulfide (GSSG) transport showed a biphasic Line-weaver-Burk plot as a function of GSSG concentration suggesting the operation of two different processes. One phase had a high affinity for GSSG and a low transport velocity. Most active at acidic pH and at 25°C, this transport activity was easily lost during the storage of vesicles at 4°C. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg-ATP was 0.63 mM; guanosine triphosphate (GTP) substituted for ATP gave a 340% stimulation of transport activity. Neither dithiothreitol nor thiol reagents affected this transport process. The other phase had a low affinity for GSSG and a high transport velocity. Most active at pH 7.2 and 37°C, this transport activity was stable during storage of vesicles at 4°C for several days. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg-ATP was 1.25 mM; GTP substituted with no change in activity. Dithiothreitol increased the V but did not alter the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, and thiol reagents inhibited the transport. These findings suggest that there are two independent transfer processes for GSSG in human erythrocytes.     

Cotransport of phosphate and sodium by yeast

Phosphate uptake by yeast at pH 7.2 is mediated by two mechanisms, one of which has a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 30 μM and is independent of sodium, and a sodium-dependent mechanism with a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 0.6 μM, both $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values with respect to monovalent phosphate. The sodium-dependent mechanism has two sites with affinity for Na⁺, with affinity constants of 0.04 and 29 mM. Also lithium enhances phosphate uptake; the affinity constants for lithium are 0.3 and 36 mM. Other alkali ions do not stimulate phosphate uptake at pH 7.2. Rubidium has no effect on the stimulation of phosphate uptake by sodium.Phosphate and arsenate enhance sodium uptake at pH 7.2. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of this stimulation with regard to monovalent orthophosphate is about equal to that of the sodium-dependent phosphate uptake.The properties of the cation binding sites of the phosphate uptake mechanism and those of the phosphate-dependent cation transport mechanism have been compared. The existence of a separate sodium-phosphate cotransport system is proposed.     

Carnitine movement across muscle cell membranes. Studies in isolated rat muscle

l-Carnitine uptake and exodus was studied in rat extensor digitorum longus muscle in vitro. A saturable transport process was observed, which had an apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 60 μM and $\text{V}$ of 22 nmol/h per g tissue. Transport was inhibited by 2,4-dinitrophenol, sodium azide, anaerobiosis, ouabain, and sodium ion depletion. Analogs of l-carnitine containing a quarternary ammonium group were found to inhibit uptake (d-carnitine, $\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 400 μ}\text{M}$ γ-butyrobetaine, $\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 60 μ}\text{M}$, choline chloride, $\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 14}\text{mM}$), while those not containing this functional group (γ-aminobutyrate, d,l-β-hydroxybutyrate) had no significant effect at concentrations 100 times the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of l-carnitine. Carnitine exodus from rat extensor digitorum longus muscle consisted of two phases. The rapid initial phase was attributed to leakage of l-carnitine from damaged muscle fibers, as it proceeded at nearly the same rate at 0° and 37°C, and leveled off to a rate of near zero after 1 h of incubation in vitro. The quantitatively more important phase of exodus showed a latency of 1–2 h and then proceeded at a linear rate of 40–45 nmol/h per g tissue. The results of this study support the contention that l-carnitine is taken up by a carrier-mediated, active transport system in rat extensor digitorum longus muscle. Functionally, the transport system for uptake is distinct from the process by which carnitine is lost from this muscle.     

Localization of the Na+-sugar cotransport system in a kidney epithelial cell line (LLC PK1)

Studies of the localization of the Na⁺-dependent sugar transport in monolayers of LLC PK₁ cells show that the uptake of a methyl α-d-glucoside, a nonmetabolizable sugar which shares the glucose-galactose transport system, occurs mainly from the apical side of the monolayer. Kinetics of [³H]phlorizin binding to monolayers of LLC PK₁ cells were also measured. These studies demonstrate the presence of two distinct classes of receptor sites. The class comprising high affinity binding sites had a dissociation constant ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}$) of 1.2 μM and a concentration of high affinity receptors of 0.30 μmol binding sites per g DNA. The other class involving low affinity sites had a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of 240 μM with the number of binding sites equal to 12 μmol/g DNA. Phlorizin binding at high affinity binding sites is a Na⁺-dependent process. Binding at the low affinity sites on the contrary is Na⁺-independent. The mode of action of Na⁺ on the high affinity binding sites was to increase the dissociation constant without modifying the number of binding sites. The Na⁺ dependence and the matching of $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ for high affinity binding sites with the $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ of phlorizin for the inhibition of methyl α-d-glucoside strongly suggest that the high affinity phlorizin binding site is, or is part of the methyl α-d-glucoside transport system. Binding studies from either side of the monolayer also show that the binding of phlorizin at the Na⁺ dependent high affinity binding sites occurs mainly from the apical rather than the basolateral side. The specific location of the Na⁺-dependent sugar transport system in the apical membrane of LLC PK₁ cells is, therefore, another expression of the functional polarization of epithelial cells that is retained under tissue culture condition. In addition, since this sugar transport almost disappears after the cells are brought into suspension, it can be used as a marker to study the development of the apical membrane in this cell line.     

The effects of vanadate on calcium transport in dialyzed squid axons Sidedness of vanadate-cation interactions

(1) Vanadate (VO₃^?) fully inhibits the ATP-dependent uncoupled Ca efflux (Ca pump) in dialyzed squid axons. (2) Vanadate inhibits with high affinity. The mean apparent affinity ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) obtained was 7 μM. (3) Inhibition by vanadate is dependent on Ca_o. External Ca lead to a release of the inhibitory effect. ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 3}\text{mM}$). This antagonic effect can be reverted by increasing the vanadate concentration. Internal K⁺ increases the affinity of the intracellular vanadate binding site. External K⁺ has no effect on the inhibition. (4) Vanadate has no effect on the Na_o-dependent Ca efflux component (forward Na-Ca exchange) in the absence of ATP. In axons containing ATP vanadate modified this component.     

Regulation of glutamate synthase from Bacillussubtilis by Glutamine

Glutamate synthase, an important enzyme in the assimilation of ammonia, was measured in cultures of $\text{Bacillus}\text{subtilis}$ grown with different nitrogen sources. An attempt was made to correlate the specific activity to the intracellular levels of five metabolites of glutamate metabolism: aspartate, glutamate, glutamine, alanine and $\text{NH}{}_4{}^{\mathrm{+}}$. An inverse relationship was found between the activity of glutamate synthase and the pool level of glutamine. We propose that the intracellular concentration of glutamine is an important element in controlling the level of glutamate synthase.     

Inhibition of anion transport in the red blood cell by anionic amphiphilic compounds I. Determination of the flufenamate-binding site by proteolytic dissection of the band 3 protein

Flufenamate, a non-steroidal anti-inflammatory drug, is a powerful inhibitor of anion transport in the human erythrocyte ($\text{I}{}_{50}\text{= 6·10}{}^{\mathrm{?7}}\text{M}$). The concentration dependence of the binding to ghosts reveals two saturable components. [¹⁴C]Flufenamate binds with high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}_1\text{= 1.2·10}{}^{\mathrm{?7}}\text{M}$) to 8.5·10⁵ sites per cell (the same value as the number of band 3 protein per cell); it also binds, with lower affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}_2\text{= 10}{}^{\mathrm{?4}}\text{M}$) to a second set of sites (4.6·10⁷ per cell). Pretreatment of cells with 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), a specific inhibitor of anion transport, prevents [¹⁴C]flufenamate binding only to high affinity sites. These results suggest that high affinity sites are located on the band 3 protein involved in anion transport. Extracellular chymotrypsin and pronase at low concentration cleave the 95 kDa band 3 into 60 kDa and 35 kDa fragments without affecting either anion transport or [¹⁴C]flufenamate binding. Splitting by trypsin at the inner membrane surface of the 60 kDa chymotryptic fragment into 17 kDa transmembrane fragment and 40 kDa water-soluble fragment does not affect [¹⁴C]flufenamate binding. In contrast degradation at the outer membrane surface of the 35 kDa fragment by high concentration of pronase or papain decreases both anion transport capacity and number of high affinity binding sites for [¹⁴C]flufenamate. Thus it appears that 35 kDa peptide is necessary for both anion transport and binding of the inhibitors and that the binding site is located in the membrane-associated domain of the band 3 protein.     

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.     

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.     

Thiophosphorylation of (Na + K+)-ATPase yields an ADP-sensitive phosphointermediate

(1) Treatment of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ from rabbit kidney outer medulla with the $\text{γ-}{}^{35}\text{S}$ labeled thio-analogue of ATP in the presence of $\text{Na}{}^{\mathrm{+}}\text{+ Mg}{}^{\mathrm{2+}}$ and the absence of K⁺ leads to thiophosphorylation of the enzyme. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for [γ-S]ATP is 2.2 μM and for Na⁺ 4.2 mM at 22°C. Thiophosphorylation is a sigmoidal function of the Na⁺ concentration, yielding a Hill coefficient $\text{n}\text{H}\text{= 2.6}$. (2) The thio-analogue ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 35\; \mu M</mtext>}}$) can also support overall $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity, but $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ at 37°C is only $\text{1.3 γ}\text{mol}\text{· (mg protein)}{}^{\mathrm{?}}\text{·}$ h^?1 or 0.09% of the specific activity for ATP ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 0.43\; mM</mtext>}}$). (3) The thiophosphoenzyme intermediate, like the natural phosphoenzyme, is sensitive to hydroxylamine, indicating that it also is an acylphosphate. However, the thiophosphoenzyme, unlike the phosphoenzyme, is acid labile at temperatures as low as 0°C. The acid-denatured thiophosphoenzyme has optimal stability at pH 5–6. (4) The thiophosphorylation capacity of the enzyme is equal to its phosphorylation capacity, indicating the same number of sites. Phosphorylation by ATP excludes thiophosphorylation, suggesting that the two substrates compete for the same phosphorylation site. (5) The (apparent) rate constants of thiophosphorylation (0.4 s^?1 vs. 180 s^?1), spontaneous dethiophosphorylation (0.04 s^?1 vs. 0.5 s^?1) and K⁺-stimulated dethiophosphorylation (0.54 s^?1 vs. 230 s^?1) are much lower than those for the corresponding reactions based on ATP. (6) In contrast to the phosphoenzyme, the thiophosphoenzyme is ADP-sensitive (with an apparent rate constant in ADP-induced dethiophosphorylation of 0.35 s^?1, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$$\text{ADP = 48 μM at 0.1 mM ATP}$) and is relatively K⁺-insensitve. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for K⁺ in dethiophosphorylation is 0.9 mM and in dephosphorylation 0.09 mM. The thiophosphoenzyme appears to be for 75–90% in the ADP-sensitive E₁-conformation.     

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.     

Peptide receptors in human lung tumor cells in culture: vasoactive intestinal peptide (VIP) and secretin interaction with the Calu-1 and SW-900 cell lines

We have investigated the interaction of VIP and secretin with two human lung carcinoma cell lines in cultures, SW-900 and Calu-1. ¹²⁵I-labeled VIP binds to and is inactivated by SW-900 and Calu-1 cells in a time- and temperature-dependent manner. The rates of binding and of inactivation were higher at 30°C than at 15°C. At equilibrium, native VIP competitively inhibited the binding of ¹²⁵I-VIP in the 10^?10?10^?7M range, half-maximal inhibition being observed at 1.2 nM in SW-900 cells and at 1.1 nM VIP in Calu-1 cells. Scatchard analysis indicated two classes of binding sites with similar characteristics in both cell lines. SW-900 cells have 27 600 sites with a high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 0.34}\text{nM}$) and 1062 000 sites with a low affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 61.4}\text{nM}$). Calu-1 cells have 36 300 sites with a high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 0.33}\text{nM}$) and 1148 000 sites with a low affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 78.6}\text{nM}$). Secretin inhibited tracer binding but with a 5000 times lower potency than native VIP in both cell lines.     

Thiamine transport in thiamine-deficient rats role of the unstirred water layer

As part or a systematic study of alcoholism and thiamine absorption, the effect of diet-induced thiamine deficiency and the role of the unstirred water layer on thiamine transport were investigated. Using ³H-labeled dextran as a marker of adherent mucosal volume, jejunal uptake of ¹⁴C-labeled thiamine hydrochloride was measured, in vitro, in thiamine-deficient rats and pair-fed controls. Uptake of low thiamine concentrations (0.2 and 0.5 μM) was greater in the thiamine-deficient rats thatn in the controls. In contrast, uptake rates for high thiamine concentrations (20 and 50 μM) were similar in both groups. While $\text{1J}{}_{\max }$ was unaltered, $\text{1K}{}_{\mathrm{m}}$ was decreased in thiamine deficiency, suggesting a decrease in unstirred water layer thickness. Accordingly, the thickness of the water layer was measured in both groups of animals and correlated with $\text{1J}{}_{\max }$ and $\text{1K}{}_{\mathrm{m}}$ under unstirred and st irred conditions. Without stirring, there was no difference in $\text{1J}{}_{\max }$ between the two groups. In contrast, both $\text{1K}{}_{\mathrm{m}}$ and the water layer were reduced in the thiamine-deficient rats. With stirring, $\text{1J}{}_{\max }$ was not affected, but both $\text{1K}{}_{\mathrm{m}}$ and the water layer thickness were reduced to similar values in both groups. Reversal of thiamine deficiency resulted in the return of thiamine uptake and the unstirred water layer thickness to control values. These data support the concept of a dual system of thiamine transport and emphasize the role of the unstirred water layer as an important determinant of transport kinetics not only under physiologic situations but also in diet-induced rat thiamine deficiency, a model for a clinical pathological state. The decrease in the unstirred water layer thickness in thiamine deficiency may be also viewed as a possible adaptive mechanism to facilitate absorption of meager supplies of thiamine.     

Carnitine uptake into human heart cells in culture

The uptake of radiolabeled carnitine and butyrobetaine has been studied in human heart cells (CCL 27). The uptake of carnitine is 3–10-fold higher in heart cells than in fibroblasts ($\text{pmol}\text{· μ}\text{g DNA}{}^{\mathrm{?1}}$). The uptake of carnitine increases with temperature coefficient $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ of 1.6 in the interval 10–20° C and with a negligible uptake at 4 and 10° C. The uptake of carnitine follows Michaelis-Menten kinetics with a $\text{K}{}_{\mathrm{<mtext>M</mtext>}}$ of $\text{4.8 ± 2.2 μ}\text{M}$ and $\text{V = 8.7 ± 3.2}\text{pmol}\text{· μ}\text{g DNA}{}^{\mathrm{?1}}\text{·}\text{h}{}^{\mathrm{?1}}$. Carnitine uptake is suppressed 90% by NaF (24 mM). Butyrobetaine is taken up into heart cells to the same extent as carnitine with a $\text{K}{}_{\mathrm{<mtext>M</mtext>}}$ of 5.7–17.3 μM and $\text{V = 8.7–9.3}\text{pmol}\text{· μ}\text{g DNA}{}^{\mathrm{?1}}\text{·}\text{h}{}^{\mathrm{?1}}$. Butyrobetaine inhibits competitively the uptake of carnitine and carnitine inhibits the uptake of butyrobetaine to the same extent. No conversion of radiolabeled butyrobetaine to carnitine, or carnitine to methyl choline was observed intra- or extracellulary during incubation. These data are compatible with a selective transport mechanism for carnitine which is also responsible for the uptake of butyrobetaine.     

Energization of sulfate transport in yeast

Sulfate uptake by Saccharomyces cerevisiae is stimulated about 12-fold by preincubation of cells with 1% d-glucose or 1% ethanol. The $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ remains unchanged (0.34–0.38 mM), the $\text{J}{}_{\mathrm{<mtext>mar</mtext>}}$ increase from 18–20 to 195–230 and 170–185 nmol/min per g dry wt., respectively, after glucose and ethanol preincubation. The stimulation involves protein synthesis (it is suppressed by cycloheximide), has a half-time of 18 min and requires mitochondrial respiration (no or low effect in respiration-deficient mutants and those lacking ADP-ATP transport in mitochondria, as well as after anaerobic preincubation of the wild-type strain, and in low-phosphate cells). The presence of NH₄⁺ and some amino acids (e.g., leucine, aspartate, cysteine and methionine) depressed the stimulation while that of cationic amino acids (typically arginine and lysine) and of K⁺ increased it by 50–80%. The stimulated (i.e., newly synthesized) transport system was degraded with a half-life of about 10 min.     

Characterization of the β-adrenergic receptors of hamster white fat cells: Evidence against an important role for the α2-receptor subtype in the adrenergic control of lipolysis

The binding characteristics of the β-adrenergic antagonist, [³H]dihydroalprenolol, to hamster white adipocyte membranes were studied. This binding occurred at two classes of sites, one having high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.6±1.3}\text{nM}$) but low capacity ($\text{32±17}\text{fmol/mg}$ membrane protein) and one having low affinity but high binding capacity. While the binding at the high-affinity sites was competitively and stereoselectively displaced by both β-antagonists and β-agonists, competition at the low-affinity sites occurred only with β-antagonists and was non-stereoselective. Thus, the β-agonist (?)-isoproterenol was further used to define nonspecific binding. Under these conditions, saturation studies showed a single class of high-affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.6±0.5}\text{nM}$) binding sites with a binding capacity of $\text{53 ± 13}\text{fmol/mg}$ membrane protein (corresponding to $\text{4000 ± 980}$ sites per cell), and independent kinetic analysis provided a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ value of 1.9 nM. Competition experiments showed that these binding sites had the characteristics of a $\text{β}{}_1\text{-}\text{receptor}$ subtype, yielding $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ values in good agreement with the $\text{K}{}_{\mathrm{<mtext>act</mtext>}}$ and the $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ values found for agonist-stimulation and for antagonist-inhibition of adenylate cyclase in membranes and of cyclic AMP accumulation and lipolysis in intact cells. Furthermore, the ability of β-agonists to compete with this binding was severely depressed by p[NH]ppG. These results thus support the contention that the specific [³H]dihydroalprenolol binding sites defined as the binding displaceable by (?)-isoproterenol represent the physiologically relevant β-adrenergic receptors of hamster white adipocytes. Finally, studies of the lipolytic response of these cells to (?)-norepinephrine showed that the inhibitory effect of the $\text{α}{}_2\text{-}\text{component}$ of this catecholamine was apparent only when the effects of endogenous adenosine were suppressed, a result which argues against an important regulatory role for the $\text{α}{}_2\text{-}\text{receptors}$ in the adrenergic control of lipolysis in hamster white adipocytes.     

Partial purification and characterization of the (Ca2+ + Mg2+)-ATPase from squid optic nerve plasma membrane

A membrane fraction enriched in axolemma was obtained from optic nerves of the squid (Sepiotheutis sepioidea) by differential centrifugation and density gradient fractionation. The preparation showed an oligomycin- and NaN₃-insensitive (Ca²⁺ + Mg²⁺)-ATPase activity. The dependence of the ATPase activity on calcium concentration revealed the presence of two saturable components. One had a high affinity for calcium ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}{}^1\text{= 0.12 μ}\text{M}$) and the second had a comparatively low affinity ($\text{K}{}^2{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 49.5 μ}\text{M}$). Only the high-affinity component was specifically inhibited by vanadate (K₁ = 35 μM). Calmodulin (12.5 μ/ml) stimulated the (Ca²⁺ + Mg²⁺)-ATPase by approx. 50%, and this stimulation was abolished by trifluoperazine (10 μM). Further treatment of the membrane fraction with 1% Nonidet P-40 resulted in a partial purification of the ATPase about 15-fold compared to the initial homogenate. This (Ca²⁺ + Mg²⁺)-ATPase from squid optic nerve displays some properties similar to those of the uncoupled Ca²⁺-pump described in internally dialyzed squid axons, suggesting that it could be its enzymatic basis.     

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.