Metabolism and uptake of L-pipecolic acid by brain and heart

Metabolism and uptake of $\text{L}$-[1-¹⁴C]pipecolate was studied in the rat through tail vein injection at low (30 μg/kg) and high (30 mg/kg) doses. No radioactive compound other than $\text{L}$-pipecolate was detected in the brain or heart samples 0.5 to 60 min after injection. The contents of $\text{L}$-pipecolate in the brain dropped rapidly to reach a plateau value 2 min after injection both in the low and high dose experiments (from 0.06 to 0.05 and from 86 to 55 nmole/g brain, respectively). Similar results were observed for the heart except that the heart had $\text{L}$-pipecolate contents 2–3 fold higher than the brain at every time interval. The influx of $\text{L}$-pipecolate to the brain, as measured by the plasma/brain ratio of its contents, was 3 fold lower than the heart at each sampling point throughout the course of measurement for both dosages. The influx of $\text{L}$-pipecolate from the plasma to the heart reached an equilibrium, i.e., plasma/heart = 1, 60 min after injection for both dosages; the plasma to brain ratio was 3 at 60 min. These results indicate that there is a blood-brain transport barrier for $\text{L}$-pipecolate in the rat, which may account for the differences in neuronal effects of $\text{L}$-pipecolate when administered intracerebrally or intraperitoneally.     

Suppression of iron uptake deficiency in Escherichia coli K-12 by loss of two major outer membrane proteins.

A novel iron uptake system was observed in pseudorevertants of $\text{Escherichia}$,$\text{coli}$ strains defective in ferrienterochelin transport. The new system is unique in that it is an active transport system that does not utilize any known siderophore. Acquisition of the new uptake system occurs concomitantly with the loss of two major outer membrane proteins (b and c) believed to function as structural components of transmembrane pores.     

On the transport of sugars and amino acids by newborn kidney: use of isolated proximal tubule

While the newborn Sprague-Dawley rat has evidence of hyperglycinuria without glycosuria transport studies employing renal cortical slices $\text{in}$$\text{vitro}$ have shown paradoxically no impairment of glycine uptake by tubule cells but an inability to actively transport sugars. In order to eliminate the architecture of the cortical slice as a complicating factor in measuring cellular uptake isolated proximal tubule fragments have been prepared from newborn rats and the cellular uptake of glycine and α-methyl-D-glucoside has been measured. The entry of glycine into the cells of newborn tubules is similar to that in adult tubules and confirms data obtained with slices. On the other hand, concentrative uptake of glucoside though impaired in newborn tubules is easily demonstrable. Isolated tubules of the newborn should provide a model $\text{in}$$\text{vitro}$ system to assess the changing characteristics of sugar transport during development.     

Differences in uncoupling effects associated with the uptake of lactose and dansyl-galactoside in Escherichia coli membrane: Active transport versus specific binding

Cytoplasmic membrane vesicles isolated from Escherichia coli take up dansyl-galactoside, a fluorescent competitive inhibitor of lactose transport, to much lower levels than lactose. An initial interpretation, based on the study of the fluorescent changes accompanying the energy-dependent uptake, was that it represented a one-to-one specific binding to the lac carrier protein which was not followed by transport. Recently, on the basis of a new estimation of the number of lac carrier in the membrane, it has been advanced that the uptake of dansyl-galactoside represents a nonspecific binding on the inner surface of the membrane following transport. We discriminate between the two interpretations by comparing the effects of lactose and dansyl-galactoside uptake on the electrochemical gradient of protons ($\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{H}}\text{+}$), generated by the oxidation of substrates, and on the uptake of proline. Indeed, it is known that the rate of lactose transport is such that it leads, as a consequence of the lactose/H⁺ symport, to an observable decrease of $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{H}}\text{+}$, and secondary to this decrease to an inhibition of the uptake of proline transported at much lower rate. We show that the rates of uptake of lactose and dansyl-galactoside by the membrane vesicles are similar; yet the uptake of dansyl-galactoside does not lead to the uncoupling effects which are associated with the uptake of lactose. We discuss the possible reasons for the absence of this uncoupling effect, and we conclude that our data are incompatible with the notion that the energy-dependent uptake of dansyl-galactoside is associated with an active transport involving a dansyl-galactoside/H⁺ symport. On the contrary, the data substantiate the initial interpretation that the energy-dependent uptake of dansyl-galactoside reflects the binding to the lac carrier not followed by transport.     

Endotoxin-induced alterations in glucose transport in isolated adipocytes

2-Deoxyglucose and $\text{3-O-}\text{methyglucose}$ were used to assess endotoxin-induced changes in glucose transport in rat adipocytes. 6 h after Escherichia coli endotoxin injection insulin-stimulated 2-deoxyglucose uptake was significantly depressed ($\text{V}\text{decreased}\text{, K}{}_{\mathrm{<mtext>m</mtext>}}\text{unaltered}$), phosphorylation of 2-deoxyglucose was seemingly unimpaired; basal 3-methylglucose entry was significantly increased, insulin-stimulated uptake was unaltered. Insulin significantly reduced $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ in control and endotoxin-treated cells. Cytochalasin B-insensitive uptake of both 2-deoxyglucose and 3-methylglucose, a small fraction of total transport, increased significantly in endotoxic cells. Endotoxin reduced spermine- and insulin-stimulated 2-deoxyglucose uptake to a similar extent. Results are consistent with the hypotheses that (1) a site of endotoxin-induced insulin resistance is at the cell membrane level and may reflect a decrease in number or activity of effective carrier units, rather than alterations in affinity, (2) endotoxin does not compromise the hexokinase system, (3) the cell membrane-localized effect of endotoxin on hexose transport is not necessarily mediated by the insulin receptor and (4) the entry of 2-deoxyglucose and 3-methylglucose may involve two separate transport systems.     

Distinct mechanisms of hypoxanthine and inosine transport in membrane vesicles isolated from Chinese hamster ovary and Balb 3T3 cells

Both enzyme-mediated group translocation and facilitated diffusion have been proposed as mechanisms by which mammalian cells take up purine bases and nucleosides. We have investigated the mechanisms for hypoxanthine and inosine transport by using membrane vesicles from Chinese hamster ovary cells (CHO), Balb/c 3T3 and SV3T3 cells prepared by identical procedures. Uptake mechanisms were characterized by analyzing intravesicular contents, determining which substrates could exchange with the transport products, assaying for hypoxanthine phosphoribosyltransferase activity, and measuring the stimulation of uptake of hypoxanthine by phosphoribosyl pyrophosphate ($\text{P}\text{Rib}\text{-PP}$).We found that the uptake of hypoxanthine in Balb 3T3 vesicles was stimulated 3–4-fold by $\text{P}\text{Rib}\text{-PP}$. The intravesicular product was predominantly IMP. The hypoxanthine phosphoribosyltransferase activity copurified with the vesicle preparation. These results suggest the possible involvement of this enzyme in hypoxanthine uptake in 3T3 vesicles. In contrast to the 3T3 vesicles, CHO vesicles prepared under identical procedures did not retain hypoxanthine phosphoribosyltransferase activity and did not demonstrate $\text{P}\text{Rib}\text{-PP}$-stimulated hypoxanthine uptake. The intravesicular product of hypoxanthine uptake in CHO vesicles was hypoxanthine. These results and data from our kinetic and exchange studies indicated that CHO vesicles transport hypoxanthine via facilitated diffusion. An analogous situation was observed for inosine uptake; CHO vesicles accumulated inosine via a facilitated diffusion mechanism, while in the same experiments SV3T3 vesicles exhibited a purine nucleoside phosphorylase-dependent translocation of the ribose moiety of inosine.     

The role of microfilaments and of microtubules in taurocholate uptake by isolated rat liver cells

The role of microfilaments and microtubules on bile salt transport was studied by investigating the influence of a microfilament and a microtubule inhibitor, cytochalasin B and colchicine, respectively, on taurocholate uptake by isolated hepatocytes in vitro. Hepatocytes were prepared by the enzyme perfusion method and [¹⁴C]taurocholate uptake velocity was determined by a filtration assay. Taurocholate uptake obeyed Michaelis-Menten kinetics, maximal uptake velocity and apparent half-saturation constants averaging $\text{0.87 ±}\text{SD}\text{0.05}\text{nmol}\text{· s}{}^{\mathrm{?1}}\text{· 10}{}^{\mathrm{?6}}\text{cells}$ and $\text{10.9 ± 1.8 μ}\text{M}$, respectively. Cytochalasin B ($\text{4.2–420 μ}\text{M}$) inhibited taurocholate uptake in a competitive fashion; $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ being $\text{33 ± 7 μ}\text{M}$. At concentrations above 100 μM the compound decreased ³⁶Cl membrane potential and intracellular K⁺ concentration. Other parameters of cell viability were not affected by cytochalasin B. Colchicine (0.1–1.0 mM), by contrast, inhibited taurocholate uptake non-competitively, $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ being $\text{0.47 ± 0.07}\text{mM}$. The inhibition brought about by colchicine was considerably smaller than that induced by cytochalasin B. None of the parameters of cell viability tested was affected by colchicine. These results suggest that microfilaments may be involved in the carrier-mediated hepatocellular transport of bile salts. This could, at least in part, account for cytochalasin B-induced cholestasis. The contribution of the microtubular system, if any, is less important quantitatively. The mechanisms whereby these two components of the cytoskeleton partake in bile salt transport remain to be elucidated.     

Methionine transport in S37 cells. Substrate-dependent function of amino acid transport system A in exchange processes

Methionine had been observed to interact with two principal transport systems for amino acids in mammalian cells, the A and L systems. The present study of methionine transport and of exchange processes through system A arose in the course of a study to define the specificity of a transinhibition effect caused by cysteine.Methionine uptake through two transport systems in the S37 cell was confirmed by the occurrence of a biphasic double-reciprocal plot for labeled methionine uptake. Preloading cells with methionine stimulated labeled histidine uptake through both systems A and L. Efflux of labeled methionine from cells was stimulated by histidine in a biphasic manner, so that both systems A and L can be used for exchange when methionine is the intracellular amino acid. Aminocycloheptanecarboxylic acid elicited exchange efflux of labeled methionine only through system L. α-Aminoisobutyric acid and $\text{N-}\text{methyl}\text{-α-}\text{aminoisobutyric acid}$ both stimulated efflux of labeled $\text{N-}\text{methyl}\text{-α-}\text{aminoisobutyric acid}$ from S37 cells. These findings are interpreted a showing that transport system A is capable of functioning as an exchange system depending upon the identity of intracellular and extracellular substrates available.     

Cysteine as a system-specific substrate for transport system ASC in rat hepatocytes.

The rapid transport of $\text{L}$-cysteine into isolated rat hepatocytes escapes detectable inhibition by 2-(methylamino)-isobutyric acid at levels up to 50 mM. The system transporting cysteine instead is convincingly similar to the $\text{ASC}$ system described for the Ehrlich cell in structural and steric specificity and in pH sensitivity. The Na⁺-dependent uptake of 2-aminoisobutyric acid is almost evenly divided between Systems $\text{A}$ and $\text{ASC}$, showing better accommodation of its two α-methyl groups by $\text{ASC}$ than in the Ehrlich cell. The hepatocyte $\text{ASC}$ system tolerates Li⁺-for-Na⁺ substitution better than does System $\text{A}$, although the tolerance depends on amino acid structure. Adaptive regulation and insulin and glucagon stimulation were not seen under conditions producing these effects for System $\text{A}$.     

Ribosomal proteins L7/L12 localized at a single region of the large subunit by immune electron microscopy.

Ribosomal proteins $\text{L}\text{7}\text{L}\text{12}$ have been mapped by immune electron microscopy. These multiple copy proteins are located at a single region extending from the large subunit, known as the $\text{L}\text{7}\text{L}\text{12}$ stalk. The $\text{L}\text{7}\text{L}\text{12}$ stalk is approximately 100 Å long, about 40 Å wide and extends at an angle of approximately 50 ° from one side of the central protuberance of the large subunit. In the monomeric 70 S ribosome, the portion of the $\text{L}\text{7}\text{L}\text{12}$ stalk proximal to the 50 S subunit is located in the vicinity of the 30 S-50 S interface.Anti-$\text{L}\text{7}\text{L}\text{12}$ antibody binding to the stalk was shown to be solely dependent upon the presence of $\text{L}\text{7}\text{L}\text{12}$ by the following experiments. Sucrose gradient analysis was used to demonstrate that large subunits depleted of $\text{L}\text{7}\text{L}\text{12}$ were unable to bind anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies and that re-incorporation of $\text{L}\text{7}\text{L}\text{12}$ restored the ability of $\text{L}\text{7}\text{L}\text{12}$-depleted cores to react with anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies. Anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies pre-absorbed with $\text{L}\text{7}\text{L}\text{12}$ did not react with 50 S subunits.Anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies used in these experiments reacted only with the $\text{L}\text{7}\text{L}\text{12}$ stalk and with no other region of the subunit. This was shown by electron microscopy and by immune electron microscopy in the following ways. Electron microscopy of 50 S subunits, $\text{L}\text{7}\text{L}\text{12}$-depleted 50 S cores, and reconstituted 50 S subunits was used to demonstrate that stripping removes the $\text{L}\text{7}\text{L}\text{12}$ stalk from more than 95% of the subunits, and that re-incorporation of $\text{L}\text{7}\text{L}\text{12}$ into depleted cores restores the $\text{L}\text{7}\text{L}\text{12}$ stalk. Double-labelling experiments, using monomeric subunits with two or more attached anti-$\text{L}\text{7}\text{L}\text{12}$ immunoglobulins, were used to demonstrate, independently of 50 S subunit morphology, that $\text{L}\text{7}\text{L}\text{12}$ are located only on the $\text{L}\text{7}\text{L}\text{12}$ stalk.     

The effect of diamide and glutathione on the uptake of α-methyl-d-glucoside by slices of rat kidney cortex

The uptake of α-methyl-d-glucoside was stimulated in slices of rat kidney cortex by pretreatment with reduced glutathione. Diamide, an oxidizing agent with high specificity for GSH, caused an inhibition of α-methyl-d-glucoside uptake. These effects appeared to be related specifically to GSH, since dithiothreitol and mercaptoethanol did not increase α-methyl-d-glucoside uptake, and were not as effective as GSH in reversing the effects of diamide. GSH and diamide had no effect on the uptake of another sugar analog, $\text{3-O-}\text{methylglucose}$, which is not actively transported. Kinetic studies indicated that GSH increased the apparent $\text{V}$ without affecting $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. The results are discussed in terms of the possible role of GSH in the process of sugar transport.     

Uncoupler and anaerobic resistant transport of phosphate in Escherichia coli

Active transport of inorganic phosphate into whole cells of a strain (AB3311) derived from $\text{Escherichia coli}$ K12 was found to be partially resistant to 50 μM carbonyl cyanide $\text{m}$-chlorophenyl hydrazone (CCCP), a powerful uncoupler of oxidative phosphorylation. The presence of 10 mM dithiothreitol (DTT) before the addition of CCCP completely prevented the inhibition of phosphate uptake caused by the uncoupler. The addition of DTT to the CCCP-inhibited system restored phosphate uptake to the control rate even when added 5 min after the phosphate transport assay was started. This uncoupler resistant transport is insensitive to anaerobiosis, or the addition of 10 mM KCN which reduces oxygen consumption to less than 1% that of aerobic controls. Additional studies of transport in a mutant (CBT302) deficient in membranebound Ca²⁺-, Mg²⁺-ATPase activity also demonstrated the retention of appreciable inorganic phosphate uptake under anaerobic conditions.     

Determination of the kinetic constants of fructose transport and phosphorylation in the perfused rat liver

The kinetics of fructose uptake was determined in perfused rat liver during steady-state fructose elimination. On the basis of the corresponding values of fructose concentration in the affluent and in the effluent medium, and the fructose and ATP concentration in biopsies, the kinetics of membrane transport and intracellular phosphorylation in the intact organ was calculated according to a model system. Carrier-mediated fructose transport has a high $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ (67 mM) and $\text{V}$ (30 μmoles · min^?1 ·g^?1). The calculated kinetic constants of the intracellular phosphorylation were compared with values obtained with an acid-treated rat liver high speed supernatant (values given in parentheses). $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ with fructose 1.0 mM (0.7 mM), $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ with ATP 0.54 mM (0.37 mM), $\text{V}$ 10.3 μmoles · min^?1 · g^?1 (10.1 μmoles · min^?1 · g^?1, calculated on the basis of the highest measured rate of fructose uptake correcting the ATP concentration to saturating values). The kinetics of fructose uptake reveals that at Physiological fructose concentrations the membrane transport limits the rate of fructose uptake, thus protecting the liver from severe depletion of adenine nucleotides.     

Properties of carnitine transport in rat kidney cortex slices

The properties of carnitine transport were studied in rat kidney cortex slices. Tissue: medium concentration gradients of 7.9 for L-[methyl-¹⁴C]carnitine were attained after 60-min incubation at 37°C in 40 μM substrate. L- and D-carnitine uptake showed saturability. The concentration curves appeared to consist of (1) a high-affinity component, and (2) a lower affinity site. When corrected for the latter components, the estimated K_m for L-carnitine was 90 μM and $\text{V = 22}\text{nmol/min}$ per ml intracellular fluid; for D-carnitine, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 166}\text{μM}$ and $\text{V = 15}\text{nmol/min}$ per ml intracellular fluid. The system was stereospecific for L-carnitine. The uptake of L-carnitine was inhibited by (1) D-carnitine, γ-butyrobetaine, and (2) acetyl-L-carnitine. γ-Butyrobetaine and acetyl-L-carnitine were competitive inhibitors of L-carnitine uptake. Carnitine transport was not significantly reduced by choline, betaine, lysine or γ-aminobutyric acid. Carnitine uptake was inhibited by 2,4-dinitrophenol, carbonyl cyanide $\text{m-}\text{chlorophenylhydrazone}$, N₂ atmosphere, KCN, $\text{N-}\text{ethylmaleimide}$, low temperature (4°C) and ouabain. Complete replacement of Na⁺ in the medium by Li⁺ reduced L- and D-carnitine uptake by 75 and 60%, respectively. Complete replacement of K⁺ or Ca²⁺ in the medium also significantly reduces carnitine uptake. Two roles for the carnitine transport system in kidney are proposed: (1) a renal tubule reabsorption system for the steady-state maintenance of plasma carnitine; and (2) maintenance of normal carnitine levels in kidney cells, which is required for fatty acid oxidation.     

Inhibitory effect of unconjugated bilirubin on p-aminohippurate transport in rat kidney cortex slices

(1) The effects of unconjugated bilirubin on the accumulation of $\text{p-}\text{aminohippurate}$, kinetics of $\text{p-}\text{aminohippurate}$ uptake, the efflux of pre-accumulated $\text{p-}\text{aminohippurate}$ and water and electrolyte distribution were investigated in the rat kidney cortical slice. (2) The addition of unconjugated bilirubin to the incubation medium decreased the 60 min slice-to-medium concentration ratio of $\text{p-}\text{aminohippurate}$. (3) The decrease in $\text{p-}\text{aminohippurate}$ accumulation by unconjugated bilirubin was found to be more pronounced by increasing the concentration of pigment in the medium. (4) The rate of uptake of $\text{p-}\text{aminohippurate}$ as a function of $\text{p-}\text{aminohippurate}$ concentration differed in aerobiosis and anaerobiosis, and unconjugated bilirubin decreased only the uptake of $\text{p-}\text{aminohippurate}$ in aerobic conditions. (5) The efflux of pre-accumulated $\text{p-}\text{aminohippurate}$ decreased when unconjugated bilirubin concentration in the medium was low (10–20 μM) but the efflux increased when the concentration of pigment was much higher (100 μM). (6) The addition of unconjugated bilirubin to the medium (40–100 μM) increased intracellular sodium and total tissue water content, and decreased intracellular potassium and oxygen consumption of tissue. However the slices incubated with low concentration of pigment (20 μM) did not exhibit significative changes in cellular functional parameters. (7) These findings suggest that unconjugated bilirubin impairs $\text{p-}\text{aminohippurate}$ transport by a complex mechanism that might involve binding of pigment to sites necessary for anion transport, although effects related to pigment toxicity or to its oxidative decomposition are not excluded.     

Effect of N-ethylmaleimide on leucine transport in chang liver cells. I. Stimulatory effect on Na+-independent transport at low concentrations of leucine

Pretreatment of Chang liver cells with $\text{N-}\text{ethylmaleimide}$ (0.5 or 1 mM) stimulated Na⁺-independent uptake of leucine at low concentrations (?1 mM). The stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on the uptake of leucine measured in Na⁺-replete medium was completely blocked by the addition of $\text{b-2-}\text{aminobicyclo}\text{[2,2,1]}\text{heptane}\text{-2-}\text{carboxylate}$ (5 mM), which shows that the L system participates in the stimulation. The Na⁺-dependent uptake of glycine was depressed by $\text{N-}\text{ethylmaleimide}$ pretreatment. The stimulation of the Na⁺-independent component of leucine uptake continued for at least 30 min after $\text{N-}\text{ethylmaleimide}$ treatment, while the inhibition of glycine uptake was progressive with time and the Na⁺-dependent uptake of leucine became depressed later, after the treatment. It has been demonstrated that treatment of cells with $\text{N-}\text{ethylmaleimide}$ is capable of increasing the Na⁺-independent influx of leucine and at the same time slightly decreasing the efflux of it. These results suggest that $\text{N-}\text{ethylmaleimide}$ attacks the Na⁺-independent system of amino acid transport at the reactive SH groups(s) of relevant protein(s) in favor of specific activation of that system in this cell.     

The uptake of (3H)dopamine by homogenates of rat corpus striatum: effects of cations

The uptake of [³H]dopamine was studied with a synaptosomal preparation of the corpus striatum. The accumulation of dopamine was found to be temperature-dependent and very rapid, but linear over time for at least 5 min. at 37°C with characteristics of saturable kinetics. The optimum concentrations for Na⁺ and K⁺ were 150–160 m$\text{M}$ and 2.5–4.8 m$\text{M}$, respectively, while uptake was progressively inhibited at concentrations of K⁺ greater than 5 m$\text{M}$. Rubidium was capable of substituting for potassium whereas cesium was a much less effective replacement. The uptake of DA was blocked by the antibiotics, valinomycin and gramicidin-D which bind K⁺ or both Na⁺ and K⁺, respectively, and thereby might interfere with the transport of cations across neuronal membranes. Similarly, ouabain which blocks the active transport of Na⁺ markedly antagonized the accumulation of DA into striatal homogenates. In contrast, tetrodotoxin which does not prevent the active transport of Na⁺, had no effect. Uptake appeared not to require Ca⁺⁺ and it was not inhibited by increasing total osmolarity to 400 mos$\text{M}$. In general, the cationic requirements for DA-uptake in striatal tissue and its responses to several inhibition of ionic transport, do not appear to be greatly different from those reported for NE with synaptosomes prepared from whole brain.     

Gel electrophoretic studies on ribosomal proteins L7L12 and the Escherichia coli 50 s subunit

Three forms of the 50 S ribosomal subunit of Escherichia coli have been separated by agarose/acrylamide gel electrophoresis. The slowest migrating form, S-50 S, corresponded to native 50 S subunits and contained four copies of proteins $\text{L}\text{7}\text{L}\text{12}$. Removal of the four copies of this protein produced a more rapidly migrating form, M-50 S. The M-50 S form was then converted to the fastest migrating form, F-50 S, by removal of additional proteins, including L10 and L11. A one-step removal of a pentameric complex of four copies of $\text{L}\text{7}\text{L}\text{12}$ plus L10 converted the S-50 S subunit directly to the F-50 S subunit. These proteins recombined specifically with the appropriate protein-deficient 50 S subunit at 3 °C to reform the S-50 S subunit, i.e. the M-50 S subunit was converted back to the S-50 S form by the addition of purified proteins $\text{L}\text{7}\text{L}\text{12}$; and the F-50 S subunit bound the pentameric complex of $\text{L}\text{7}\text{L}\text{12}$ and L10 to form S-50 S. The binding of the pentameric complex, isolated by glycerol gradient centrifugation, supports the model that all four copies of proteins $\text{L}\text{7}\text{L}\text{12}$ are together in one part of the ribosome called the “$\text{L}\text{7}\text{L}\text{12}$ stalk”. Only the four copies of $\text{L}\text{7}\text{L}\text{12}$ were removed from the 50 S subunit in low salt (0.125 m-NH₄Cl) plus 50% ethanol at 0 °C. These ribosomes (in the M-50 S form) had less than 5% of the peptide-synthesizing activity of untreated control ribosomes as measured by a poly(U) translation system in vitro. Peptide-synthesizing activity was restored, upon addition of $\text{L}\text{7}\text{L}\text{12}$, back to the treated ribosomes to give 50 S subunits (S-50 S) with a full complement of four copies of $\text{L}\text{7}\text{L}\text{12}$. Antibody to proteins $\text{L}\text{7}\text{L}\text{12}$ bound only to the S-50 S subunits, producing four new bands separated by gel electrophoresis. The bands represented complexes of one, two, three and four antibodies bound to a 50 S subunit. This result was obtained using either 50 S subunits or 70 S tight couples and indicated that all four copies of $\text{L}\text{7}\text{L}\text{12}$ are either located at a single site in the $\text{L}\text{7}\text{L}\text{12}$ stalk or, much less likely, are divided between two symmetrical sites. Proteins $\text{L}\text{7}\text{L}\text{12}$ were not only accessible to their specific antibody but could also be removed from 70 S ribosomes and polyribosomes without causing their dissociation into subunits. The ribosomes and polyribosomes had an increased gel electrophoretic mobility which was reversed by addition of proteins $\text{L}\text{7}\text{L}\text{12}$.