Defective PAPS-synthesis in epiphyseal cartilage from brachymorphic mice

Activity levels of sulfotransferases, requisite for the sulfation of chondroitin sulfate proteoglycan, were measured in cell-free homogenates prepared from neonatal epiphyseal cartilage of normal C57B1/6J or homozygous brachymorphic mice. In the presence of [³⁵S]-PAPS only or [³⁵S]-PAPS plus an exogenous sulfate acceptor, comparable amounts of ${}^{35}\text{SO}{}_4{}^{\mathrm{2?}}$ were incorporated into chondroitin sulfate by the normal and mutant types of cartilage. In contrast, the mutant cartilage catalyzed the conversion of only 30% of the ${}^{35}\text{SO}{}_4{}^{\mathrm{2?}}$ into chondroitin sulfate as compared to normal mouse cartilage when synthesis was initiated from ATP and H₂³⁵SO₄. These results suggest that the production of an undersulfated proteoglycan which has previously been reported in brachymorphic mice (Orkin, R.W. $\text{et}\text{al}$. (1976) Devel. Biol. $\text{50}$, 82–94) may result from a defect in the synthesis of the sulfate donor PAPS.     

Aberrant metabolism of matrix components in neonatal fibular cartilage of brachypod (bpH) mice.

Collagen and the acid mucopolysaccharides (AMPS) were studied in the fibular cartilage of brachypod ($\text{bp}{}^{\mathrm{<mtext>H</mtext>}}\text{bp}{}^{\mathrm{<mtext>H</mtext>}}$) and normal (+/+) newborn mice. At this age the mutant fibulae are still cartilaginous and are comprised of closely packed chondrocytes, homogenous in size and shape.Brachypod fibular chondrocytes synthesized a normal cartilage-type collagen molecule but at half the normal rate. Incorporation of tryptophan indicated this was related to a depression of general protein synthesis rather than being specific for collagen. Pulse-chase experiments showed that collagen degradation over a 3-day culture period was 15% slower than normal thus accounting for the higher collagen content in mutant fibulae.AMPS synthesis in normals and brachypods was nearly equal; however, in pulse-chase experiments radioactivity could not be chased out of the mutant tissue. The failure of AMPS degradation also accounted for greater than normal quantity of AMPS in the mutant cartilage. Characterization of the AMPS led to the discovery of a small population of unsulfated chondroitin molecules in normal, but not brachypod cartilage. The importance of a coordinated metabolism of matrix products during limb development is discussed.     

Molecular structure of collagen in solution: comparison of types I,II, III and V

Physical properties of pepsin-solubilized types I, II, III and V collagen have been measured in acid solution at 10°C. Our results indicate that types I, II and III collagen molecules undergo a monomer-aggregate equilibrium in solution whereas type V molecules appear to attract each other but do not undergo a similar monomer-aggregate equilibrium. Interstitial collagen monomers (I, II and III) have molecular weights between $\text{280 × 10}{}^3\text{and 289 × 10}{}^3$, translational diffusion coefficients between $\text{0.820 × 10}{}^{\mathrm{?7}}\text{and 0.845 × 10}{}^{\mathrm{?7}}\text{cm}{}^2\text{s}{}^{\mathrm{?1}}$ and particle scattering factors at an angle of 175.5° and wavelength of 633 nm between 0.430 and 0.460. Type V collagen molecules after pepsin digestion were found to have a higher molecular weight ($\text{307 × 10}{}^3$), similar translational diffusion coefficient ($\text{0.860 × 10}{}^{\mathrm{?7}}\text{cm}{}^2\text{s}{}^{\mathrm{?1}}$) and similar particle scattering factor at 175.5° (0.440) to the interstitial collagens. Theoretical bead models are discussed and suggest that changes in the translational diffusion coefficient were less sensitive to bending motions than were changes in the particle scattering factor at 175.5°C. Bend angles of 50° were shown to increase the particle scattering factor by 5% whereas a bend angle of greater than 125° was required to increase the translational diffusion coefficient by 5%. Models developed from idealized shapes seen by electron microscopy of rotary shadowed collagen molecules agreed best with experimental laser light scattering measurements when the bend angles were less than 90°.     

Purification of cytochromes P-448 from β-naphthoflavone-treated rainbow trout

Rainbow trout were treated with β-naphthoflavone and the hepatic microsomal cytochrome P-450 solubilized with 3-[(3-cholaminopropyl)dimethylammonio]-1-propanesulfonate. Chromatography on tryptamine-Sepharose 4B gave a single cytochrome P-450 peak which was further resolved into three components by elution from DEAE-Sepharose. The two main peaks were then chromatographed on hydroxyapatite and a total of four fractions obtained. Two of these fractions had similar properties and significantly metabolized $\text{[}{}^{14}\text{C}\text{]}\text{benzo}\text{[a]}\text{pyrene}$ in a reconstituted system containing rat cytochrome P-450 reductase. This activity was inhibited by α-naphthoflavone but not by metyrapone or SKF-525A. Purified cytochromes P-448 from 3-methylcholanthrene-treated rat had similar spectral properties and activity towards $\text{[}{}^{14}\text{C}\text{]}\text{benzo}\text{[a]}\text{pyrene}$ suggesting similarities between these forms.     

Kinetic considerations for the regulation of adenosine and deoxyadenosine metabolism in mouse and human tissues based on a thymocyte model

Metabolic regulation at a branch point may be determined primarily by relative enzyme activities and affinity for common substrate. Adenosine and deoxyadenosine are both phosphorylated and deaminated and their metabolism was studied in intact mouse thymocytes. From kinetic considerations of two activities competing for a common substrate, the deamination:phosphorylation ratio, $\text{v}{}^{\mathrm{<mtext>d</mtext>}}\text{v}{}^{\mathrm{<mtext>k</mtext>}}$, at high nucleoside concentration, $\text{[}\text{S}\text{]?∞}$, is equal to $\text{V}{}^{\mathrm{<mtext>d</mtext>}}\text{V}{}^{\mathrm{<mtext>k</mtext>}}$, or 34 and 1090 for adenosine and deoxyadenosine, respectively. At low substrate concentrations, $\text{[}\text{S}\text{]?0}$, $\text{v}{}^{\mathrm{<mtext>d</mtext>}}\text{v}{}^{\mathrm{<mtext>k</mtext>}}$ is equal to $\text{V}{}^{\mathrm{<mtext>d</mtext>}}\text{K}{}^{\mathrm{<mtext>k</mtext>}}{}_{\mathrm{<mtext>m</mtext>}}\text{V}{}^{\mathrm{<mtext>k</mtext>}}\text{K}{}^{\mathrm{<mtext>d</mtext>}}{}_{\mathrm{<mtext>m</mtext>}}$, or 0.7 and 285 for adenosine and deoxyadenosine, respectively. The analysis was extended to other mouse and human tissues by measurement of adenosine kinase, deoxyadenosine kinase and adenosine deaminase activities. All tissues were found to preferentially deaminate deoxyadenosine. Three tissue types were apparent with respect to adenosine metabolism: those which preferentially phosphorylate adenosine at all concentrations, those which switch from phosphorylation to deamination between low and high adenosine concentration and those for which deamination is quantitatively important at all concentrations. Lymphoid tissues are representative of the latter category. The kinetic approach we describe offers a means of predicting nucleoside metabolism over a range of concentration which may be technically difficult to otherwise measure. The phosphorylation of adenosine and deoxyadenosine was also studied in intact thymocytes in the presence of adenosine deaminase inhibitors. The rate of deoxyadenosine phosphorylation was unaffected by coformycin or EHNA, whereas adenosine phosphorylation decreased with increasing substrate concentrations to 18% the rate in the absence of adenosine deaminase inhibitors.     

Rescue of embryonic cells homozygous for a lethal haplotype of the T/t complex: tw12

A series of recessive mutations which arrest embryonic development are located within the T/t region of chromosome 17 in the mouse. To assess whether these mutations cause death in specific differentiating cells or in all cells of the embryo, we removed the embryonic cells from normal developmental constraints and attempted to grow them ectopically in vivo and in vitro. We have succeeded in producing teratomas and teratocarcinomas by transplantation of inner cell masses from blastocysts of $\text{t}{}^{\mathrm{w12}}\text{+}$ and $\text{t}{}^{\mathrm{w12}}\text{t}{}^{\mathrm{w12}}$ genotypes. The ability of embryonic cells to grow as tumors was not affected by their genotype; 7 of the 17 tumors were homozygous for t^w12, 7 were heterozygous, and 3 could not be analyzed. Virtually all the tumors of both genotypes contained derivatives of all three germ layers. Neuroepithelial and mature nervous tissue was present in all homozygous tumors and all except one heterozygous tumor. However, no cartilage or bone was found in 5 of 5 t^w12 homozygous tumors, while both tissues were present in 3 of 4 t^w12 heterozygous tumors. This observation is compatible with the abnormalities characteristic of $\text{t}{}^{\mathrm{w12}}\text{t}{}^{\mathrm{w12}}$ embryos, which show very localized effects in nervous tissue and more general effects on bone and cartilage formation. Cells derived from homozygous tumors were capable of at least limited growth in culture and a cell line has been derived from one of them. The p63/6.9a marker protein was used to determine the presence of the t^w12 haplotype in the tumor and cultured cells. We conclude that the lethality associated with the t^w12 haplotype is due to lethality of specific cells, and not all cell types.     

DNA-binding proteins of Xenopus laevis: synthesis during oogenesis-ovulation and early embryogenesis

DNA-binding proteins of Xenopus laevis synthesized during two periods of early development (oogenesis-ovulation and early embryogenesis) were co-chromatographed on DNA-cellulose. Proteins with an affinity for DNA were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Most of the proteins eluted from DNA-cellulose with 0.6 M NaCl had mol. wts less than 40 000; some of these proteins were synthesized to a greater extent by developing embryos than by oocytes. A DNA-binding protein or group of proteins with a mol. wt of approx. 70 000 was synthesized during oogenesis-ovulation but not during embryogenesis. Differential labeling of developing embryos with $\text{[}{}^3\text{H}\text{]}\text{tryptophan}$ and $\text{[}{}^{14}\text{C}\text{]}\text{lysine}$ indicated that some of the low mol. wt DNA-binding proteins are histones. Some of these proteins also incorporated monosodium $\text{[}{}^{32}\text{P}\text{]}\text{phosphate}$. A greater fraction of the proteins synthesized by oocytes and developing embryos were bound to DNA-histone-cellulose than to DNA-cellulose. A group of low mol. wt proteins made during oogenesis-ovulation were bound more to DNA-histone-cellulose than were proteins with similar mol. wts made by developing embryos.     

Histamine,theophylline and tryptamine transport through lipid bilayer membranes

Diffusion of histamine, theophylline and tryptamine through planar lipid bilayer membranes was studied as a function of pH. Membranes were made of egg phosphatidylcholine plus cholesterol (1 : 1 mol ratio) in tetradecane. Tracer fluxes and electrical conductances were used to estimate the permeabilities to nonionic and ionic species. Only the nonionic forms crossed the membrane at a significant rate. The membrane permeabilities to the nonionic species were: histamine, $\text{3.5 · 10}{}^{\mathrm{?5}}\text{cm}\text{· s}{}^{\mathrm{?1}}$; theophylline, $\text{2.9 · 10}{}^{\mathrm{?4}}\text{cm}\text{· s}{}^{\mathrm{?1}}$; and tryptamine, $\text{1.8 · 10}{}^{\mathrm{?1}}\text{cm}\text{· s}{}^{\mathrm{?1}}$. Chemical reactions in the unstirred layers are important in the transport of tryptamine and theophylline, but not histamine. For example, as pH decreased from 10.0 to 7.5 the ratio of nonionic (B) to ionic (BH⁺) tryptamine decreased by 300-fold, but the total tryptamine permeability decreased only 3-fold. The relative insensitivity of the total tryptamine permeability to the ratio, [B]/[BH⁺], is due to the rapid interconversion of B and BH⁺ in the instirred layers. Our model describing diffusion and reaction in the unstirred layers can explain some ‘anomalous’ relationships between pH and weak acid/base transport through lipid bilayer and biological membranes.     

Elasnin,a new human granulocyte elastase inhibitor produced by a strain of Streptomyces

Elasnin, a new human granulocyte elastase inhibitor, has been isolated from $\text{Streptomyces}\text{noboritoensis}$ KM-2753. Elasnin is a neutral, lipophilic colorless and viscous oil ($\text{n}{}_{\mathrm{D}}{}^{17}\text{=1.4983}$, $\text{[α]}{}_{\mathrm{D}}{}^{18}\text{?0.9°}$, λ_max^EtOH 291 nm (ε, 7760)). The molecular formula was C₂₄H₄₀O₄ (M.W.: 392) as determined by its elemental analysis and mass spectrum. Elasnin inhibits markedly human granulocyte elastase, but is almost ineffective for pancreatic elastase, trypsin, chymotrypsin, thermolysin and papain.     

Isolation of plasma membrane vesicles,derived from transverse tubules,by selective homogenization of subcellular fractions of frog skeletal muscle in isotonic media

A new technique for isolating fragmented plasma membranes from skeletal muscle has been developed that is based on gentle mechanical disruption of selected homogenate fractions. $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-stimulated}$, Mg²⁺-dependent ATPase was used as an enzymatic marker for the plasma membrane, Ca²⁺-stimulated, Mg²⁺-dependent ATPase as a marker for sarcoplasmic reticulum, and succinate dehydrogenase for mitochondria. Cell Cell segments in an amber low-speed ($\text{800 × g}$) pellet of a frog muscle homogenate were disrupted by repeated gentle shearing with a Polytron homogenizer. Sarcoplasmic reticulum was released into the low-speed supernatant, whereas most of the plasma membrane marker remained in a white, fluffy layer of the sediment, which contained sarcolemma and myofibrils. Additional gentle shearing of the white low-speed sediment extracted plasma membranes in a form that required centrifugation at $\text{100 000 × g}$ for pelleting. This pellet, the fragmented plasma membrane fraction, had a relatively high specific activity of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-stimulated}$ ATPase compared with the other fractions, but it had essentially no Ca²⁺-stimulated ATPase activity and only a small percentage of the succinate dehydrogenase activity of the homogenate.Experimental evidence suggests that the fragmented plasma membrane fraction is derived from delicate transverse tubules rather than from the thicker, basement membrane-coated sarcolemmal sheath of muscle cells. Electron microscopy showed small vesicles lined by a single thin membrane. Hydroxyproline, a characteristic constituent of collagen and basement membrane, could not be detected in this fraction.     

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.     

Ligand-dependent reactivity of (Na+ + K+)-ATPase with showdomycin

Showdomycin inhibited pig brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na⁺, K⁺, Mg²⁺ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1)$\text{Na}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$, (2) $\text{Mg}{}^{\mathrm{2+}}$, $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{K}{}^{\mathrm{+}}$, $\text{K}{}^{\mathrm{+}}$ and none, (3) $\text{Na}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{,}\text{Na}{}^{\mathrm{+}}$, $\text{K}{}^{\mathrm{+}}\text{+}\text{Na}{}^{\mathrm{+}}$ and $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}$, (4) $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{ATP}\text{,}\text{K}{}^{\mathrm{+}}\text{+}\text{ATP}$ and $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}\text{, (5)}\text{K}{}^{\mathrm{+}}\text{+}\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$ and ATP. The highest rate was obtained in the presence of Na⁺, Mg²⁺ and ATP. The apparent concentrations of Na⁺, Mg²⁺ and ATP for half-maximum stimulation of inhibition ($\text{K}{}_{0.5}{}^{\mathrm{<mtext>s</mtext>}}$) were 3 mM, 0.13 mM and 4μM, respectively. The rate was unchanged upon further increase in Na⁺ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E₁, E₂, ES, E₁-P and E₂-P, i.e., E₂ has higher reactivity with showdomycin than E₁, while E₂-P has almost the same reactivity as E₁-P. We conclude that the reaction of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ proceeds via at least four kinds of enzyme form (E₁, E₂, E₁ · nucleotide and EP), which all have different conformations.     

Non-sterol metabolism of mevalonate in vitro: artifacts and realities.

Commercial [5-¹⁴C]mevalonate is shown to contain several radioactive impurities, which give artifactually high amounts of Hyamine bound, volatile acidic radioactivity when incubated with killed or living rat renal cortex slices, as compared with [5-¹⁴C]mevalonate purified either by liquid-liquid partition chromatography or through the enzymically generated $\text{R}\text{-5-phospho-[5-}{}^{14}\text{C]mevalonate}$ by ion-exchange chromatography. The artifactual ${}^{14}\text{CO}{}_2$ results were not diluted by incubation with increasing amounts of unlabelled mevalonate, whereas the ${}^{14}\text{CO}{}_2$ and [${}^{14}\text{C}$]cholesterol produced by rat renal cortex slices incubated with purified [5-¹⁴C]mevalonate were both diluted to the same extent by unlabelled mevalonate. It is concluded that $\text{R}\text{[5-}{}^{14}\text{C]mevalonate}$ is genuinely oxidized to ${}^{14}\text{CO}{}_2\text{in}\text{vitro}$, and that purification of substrate before its use is necessary. Production of ${}^{14}\text{CO}{}_2$ and various [${}^{14}\text{C}$]lipids from purified [5-¹⁴C]mevalonate, as a function of time and substrate concentration, by renal cortex and liver slices, is described.     

Regulation of the Ca2+-pump by calmodulin in intact cells

ATP-enriched human red cells display high rates of Ca²⁺-dependent ATP hydrolysis (16 mmol·litre cells^?1·h^?1) with a high Ca²⁺ affinity ($\text{K}{}_{0.5}\text{～0.2 μ}\text{M}$). The finding suggests a mechanism for regulation of cell Ca²⁺ levels, involving highly-cooperative stimulation of active Ca²⁺ extrusion following binding of calmodulin to the $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$.     

Spondyloepiphyseal dysplasia, chondroitin sulfate type: a possible defect of PAPS--chondroitin sulfate sulfotransferase in humans

Four patients with an unusual form of spondyloepiphyseal dysplasia excreted in the urine undersulfated chondroitin 6-sulfate (Biochem. Med. $\text{7}$, 415–423, 1973). The sera of these patients show a low activity of PAPS — chondroitin sulfate sulfotransferase, while the undersulfated chondroitin sulfate present in their urine is a much better acceptor of ${}^{35}\text{SO}{}_4$ than standard chondroitin sulfate when they are incubated with [${}^{35}\text{S}$]PAPS and normal sulfotransferases. These results suggest that in these patients the skeletal lesions are secondary to a defect in the synthesis of chondroitin sulfate involving specifically the sulfotransferase activity.     

The influence of temperature and gamma-aminobutyric acid on benzodiazepine receptor subtypes in the hippocampus of the rat

The influence of GABA on the affinity of flunitrazepam (FLU) for benzodiazepine receptor subtypes (type I and II) was studied by measurement of the competitive inhibition of [³H]FLU and [³H]propyl beta-carboline-3-carboxylate ([³H]PCC) binding. When assays were carried out at 0°C using a low concentration (0.040 nM) of [³H]PCC so that the type I receptors were selectively labelled, no significant effect of GABA (10^?4 M) on the $\text{FLU}\text{[}{}^3\text{H]}\text{PCC}$ competition curve was detected. In contrast, when assays were carried out at 0°C using [³H]FLU or a high concentration of [³H]PCC to achieve [³H]ligand receptor occupancy of both type I and type II receptors, GABA (10^?4 M) caused a significant increase in the affinity of FLU as measured by $\text{FLU}\text{[}{}^3\text{H]}\text{FLU}$ and $\text{FLU}\text{[}{}^3\text{H]}\text{PCC}$ competition experiments. Collectively, these data suggest that the influence of GABA on benzodiazepine receptor binding is mediated, primarily, by the type II receptor. It was also noted that the $\text{PCC}\text{[}{}^3\text{H]}\text{FLU}$ competition curve had a Hill coefficient of approximately 1 at 37°C as compared to the results of experiments at 0°C during which a Hill coefficient of approximately 0.7 was calculated.     

Characterization of the methotrexate transport defect in a resistant L1210 lymphoma cell line

L1210/R81 lymphoma cells are resistant to methotrexate (MTX) by virtue of a 35-fold elevation in dihydrofolate reductase and an inability to transport the folate antagonist drug effectively. In a phosphate-containing buffer there was little or no influx into the resistant cells at either 1 or 50 μm MTX. Replacement of this buffer with a 4-(2-hydroxyethyl)-1-piperazine-N′-2-ethanesulfonic acid-Mg²⁺ system resulted in an apparent influx of MTX into the resistant cells. Under these conditions, L1210/R81 cells achieved an apparent steady state at an extracellular MTX concentration of 50 μm. The apparent steady-state level of 5 nmol $\text{[}{}^3\text{H]MTX}\text{10}{}^9$ cells was well below the intracellular level of dihydrofolate reductase (45 nmol/10⁹ cells). Efflux experiments at the apparent steady state indicated that 60% of the MTX was very rapidly removed from the cells by washing. Over the range of the experiment a further 20% of the MTX effluxed more slowly ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 12}\text{min}$). The apparent influx into the resistant cells at 5 μm MTX was inhibited 13% by sodium azide (100 μm) and initially stimulated, then inhibited, by p-chloromercuriphenylsulfonic acid (100 μm). 5-Methyltetrahydrofolate (100 μm) had little effect on the process while aminopterin (100 μm) was inhibitory (68%). K_t and V values of 2 × 10^?5m and 0.31 nmol $\text{[}{}^3\text{H]MTX}\text{10}{}^9$ cells/min, respectively, were determined for the apparent influx in $\text{L}\text{1210}\text{R}\text{81}$ cells. Comparison of apparent MTX influx in the resistant cells with MTX transport in the sensitive cells indicates profound differences in the two processes. The evidence suggests that the apparent influx in the former cell line may consist of MTX binding to the cell membrane together with a small degree of MTX influx into the intracellular compartment.     

Covalent modification of chloroplast Photosystem II polypeptides by p-nitrothiophenol

Illumination of the chlorophyll $\text{a}\text{b}$ light-harvesting complex in the presence of $\text{p-}\text{nitrothio}\text{[}{}^{14}\text{C]phenol}$ caused quenching of fluorescence emission at 685 nm (77 K) relative to 695 nm and covalent modification of light-harvesting complex polypeptides. Fluorescence quenching saturated with one p-nitrothiophenol bound per light-harvesting complex polypeptide (10–13 chlorophylls); $\text{1}\text{2}$ maximal quenching occurred with one p-nitrothiophenol bound per light-harvesting complex polypeptides (190–247 chlorophylls). This result provides direct evidence for excitation energy transfer between light-harvesting complex subunits which contain 4–6 polypeptides plus 40–78 chlorophylls per complex.Illumination of chloroplasts or Photosystem II (PS II) particles in the presence of $\text{p-}\text{nitrothio}\text{[}{}^{14}\text{C]phenol}$ caused inhibition of PS II activity and labeling of several polypeptides including those of 42–48 kilodaltons previously identified as PS II reaction center polypeptides. In chloroplasts, inhibition of oxygen evolution accelerated p-nitrothiophenol modification reactions; DCMU or donors to PS II decreased p-nitrothiophenol modification. These results are consistent with the hypothesis that accumulation of oxidizing equivalents on the donor side of PS II creates a ‘reactive state’ in which polypeptides of PS II are susceptible to p-nitrothiophenol modification.     

Thymidine accumulation by choroid plexus in vitro

In vitro, the accumulation and release of [methyl-³H]thymidine ([${}^3\text{H}$]thymidine) by the isolated choroid plexus, the anatomical locus of the blood-cerebrospinal fluid barrier, was studied. With concentrations of [${}^3\text{H}$]thymidine in the medium of 1.0 μm (or greater), the choroid plexus accumulated [${}^3\text{H}$]thymidine against a concentration gradient by a process that depended on intracellular energy production but did not depend on intracellular binding or metabolism of the [${}^3\text{H}$]thymidine. This transport process was inhibited (although differentially) by various nucleosides and low temperatures but not by 2-deoxyribose or pyrimidine bases. With concentrations of less than 1.0 μm [${}^3\text{H}$]thymidine in the medium, the choroid plexus accumulated [${}^3\text{H}$]thymidine against a concentration gradient. However, the majority of the [${}^3\text{H}$]thymidine within the choroid plexus was metabolized to [${}^3\text{H}$]thymidine nucleotides at low extracellular [${}^3\text{H}$]thymidine concentrations (3 nm). This accumulation process depended, in large part, on saturable intracellular phosphorylation. Thymidine was the principal form released from choroid plexuses that had been incubated for various times in media containing concentrations of thymidine from 3 to 1.0 mm. The release of thymidine from choroid plexus was depressed by cold temperatures and a very high (2.56 mmol/kg) intracellular thymidine concentration.