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.     

Induction of a relatively fast transbilayer movement of phosphatidylcholine in vesicles. A 13C NMR study

[$\text{N-}{}^{13}\text{CH}{}_3$] Phosphatidylcholines are introduced into the outer monolayer of phosphatidylcholine vesicles with the phosphatidylcholine exchange protein from bovine liver. The transbilayer distribution of the [$\text{N-}{}^{13}\text{CH}{}_3$] phosphatidylcholine is measured with ¹³C NMR. The transbilayer movements of $\text{[N-}{}^{13}\text{CH}{}_3\text{]-}\text{dioleoyl}$ phosphatidylcholine and [$\text{N-}{}^{13}\text{CH}{}_3$] dimyristoyl phosphatidylcholine at 30°C in vesicles composed of these phosphatidylcholines are extremely slow processes with estimated half-times of days. [$\text{N-}{}^{13}\text{CH}{}_3$] Dioleoyl phosphatidylcholine introduced into dimyristoyl phosphatidylcholine vesicles migrates from the outer to the inner monolayer with a half-time of less than 12 h. The data suggest that differential changes in the lateral packing of the two monolayers might be a driving force for transbilayer transport of phospholipids.     

[3H] BOC [NLE28,31]CCK27−33, a new highly labelle ligand for CCK receptors: Binding on brain and on pancreas

Preliminary results on the binding of [³H]Boc[Nle^28,31]CCK_27^?33, designated [³H]Boc[diNle]CCK₇, on mouse brain and rat pancreas membranes are presented. This new ligand for CCK receptors possesses a high specific activity (144 Ci/mmole), and binds in a saturable manner to mouse brain (Kd = 0.49 nM, Bmax = 49 fmoles/mg protein) and rat pancreas (Kd = 4.4 nM, Bmax = 696 fmoles/mg protein). Unlabelled Boc[diNle]CCK₇ displaces [¹²⁵I]CCK₈ from its binding sites on mouse brain membranes with a high affinity, slightly superior to that of CCK₈. The order of potencies to displace [³H]Boc[diNle]CCK₇ from its binding sites was the same on mouse brain and rat pancreas: $\text{[}{}^3\text{HBoc[diNle]CCK}{}_7\text{>}\text{CCK}{}_8\text{,}\text{Boc-CCK}{}_7\text{>}\text{non-sulfated CCK}{}_8$, the pancreas binding sites being more discriminative than the brain binding sites. Thus, [³H]Boc[diNle]CCK₇ is a very promising new probe for the characterization of CCK receptors and their interaction with different CCK fragments.     

Synthesis and biological activity of [D-Trp6] chicken luteinizing hormone-releasing hormone

An agonist of chicken hypothalamic luteinizing hormone-releasing hormone (cLH-RH). [D-Trp⁶] cLH-RH, was synthesized and tested for luteinizing hormone (LH)-releasing activity using dispersed chicken anterior pituitary cells, as well as for binding to rat anterior pituitary membrane receptors. cLH-RH and mammalian LH-RH (mLH-RH) gave identical dose-response curves in stimulating chicken LH release ($\text{ED}{}_{50}\text{=1.6}$ and $\text{1.8×10}{}^{\mathrm{?9}}\text{M}$ respectively) and similar estimates of potency. The [D-Trp⁶] analogs of cLH-RH and mLH-RH stimulated LH release at lower doses ($\text{ED}{}_{50}\text{=7.0}$ and $\text{～7.0×10}{}^{\mathrm{?11}}\text{M}$ respectively) and were approximately 20-fold more potent. In contrast to the activity in the chicken bioassay, cLH-RH bound to rat anterior pituitary membrane receptors with a much lower affinity than did mLH-RH and had a relative potency of 2%. [D-Trp⁶] cLH-RH was approximately 100-fold more potent than cLH-RH in the rat receptor assay while [D-Trp⁶] mLH-RH was 28-fold more active than mLH-RH. These data demonstrate that substitution of Gly⁶ of LH-RH with D-Trp enhances the LH release from chicken pituitary cells to a similar extent to that observed in mammals, and indicate that the approaches used to produce active LH-RH analogs in mammals are likely to be applicable to birds.     

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.     

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.     

Partial reactivation of impaired immune competence in aged mice by synthetic thymus factors

The stability of mouse uterine cytosol receptor-[³H]estradiol complex was evaluated in the presence of neutral salts of the Hoffmeister series. Marked increases in the rate of dissociation of the complex were observed with the more chaotropic anions (SCN^?, $\text{ClO}{}_4{}^{\mathrm{?}}$, $\text{NO}{}_3{}^{\mathrm{?}}$, Br^?), and the effects of these ions were greater at lower temperatures, where water assumes a more rigid structure. At higher temperatures F^? and CH₃COO^?, which tend to stabilize water structure, led to retardation of the rate of dissociation of the hormone-receptor complex. There was essentially no change in steroid specificity in the presence of the markedly chaotropic salts. The perturbation of water structure adjacent to the steroid binding site is a factor to be considered in the isolation of steroid receptor complexes.     

Structure and action of heteronemertine polypeptide toxins Binding of cerebratulus lacteus toxin B-IV to axon membrane vesicles

The binding of the crustacean selective protein neurotoxin, toxin B-IV, from the nemertine Cerebratulus lacteus to lobster axonal vesicles has been studied. A highly radioactive, pharmacologically active derivative of toxin B-IV has been prepared by reaction with Bolton-Hunter reagent. Saturation binding and competition of ¹²⁵I-labeled toxin B-IV by native toxin B-IV have shown specific binding of ¹²⁵I-labeled toxin B-IV to a single class of binding sites with a dissociation constant of 5–20 nM and a binding site capacity, corrected for vesicle sidedness, of 6–9 pmol per mg membrane protein. This compares to a value of 3.8 pmol [³H]saxitoxin bound per mg in the same tissue. Analysis of the kinetics of toxin B-IV association ($\text{k}{}_{\mathrm{+1}}\text{=7.3·10}{}^5\text{M}{}^{\mathrm{?1}}\text{·s}{}^{\mathrm{?1}}$) and dissociation ($\text{k}{}_{\mathrm{?\; 1}}\text{=2·10}{}^{\mathrm{?3}}\text{s}{}^{\mathrm{?1}}$) shows a nearly identical $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of about 3 nM. There is no competition of toxin B-IV binding by purified toxin from Leiurus quinquestriatus venom while Centruroides sculpturatus Ewing toxin I appears to cause a small enhancement of toxin B-IV binding.     

A new assay for ATP sulfurylase based on differential solubility of the sodium salts of adenosine 5′-phosphosulfate and sulfate

A new assay for ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) has been devised, the key feature of which is the quantitative precipitation of the unreacted substrate as Na₂SO₄, in ethanol:water (5:1), leaving the product adenosine 5′-phosphosulfate (APS) in solution with an 80% yield. This separation procedure, coupled with the use of $\text{[}{}^{35}\text{S]SO}{}_4{}^{\mathrm{2?}}$, makes it possible to assay the synthesis of picomole amounts of [³⁵S]APS and thereby determine accurately the initial velocity of the forward reaction catalyzed by ATP sulfurylase. Extracts from tobacco cells synthesized [³⁵S]APS and a negligible amount of one unknown sulfur compond, but they did not synthesize 3′-phosphoadenosine 5′-[³⁵S]phosphosulfate (PAPS) under the assay conditions adopted, so that the ³⁵S remaining in solution after ethanol precipitation of $\text{[}{}^{35}\text{S]SO}{}_4{}^{\mathrm{2?}}$ is a valid assay of the ATP sulfurylase reaction in these extracts. This assay is simple, rapid, and suitable for assaying multiple samples. Extracts of four additional species of plants were incubated under the same conditions, and each catalyzed the synthesis of [³⁵S]APS according to this assay. The assay is suitable for use with the enzyme from any organism, provided that the product APS is not acted upon by another enzyme.     

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.     

Identification and partial characterization of plasma membrane polypeptides of Trypanosoma brucei

A plasma membrane-enriched vesicle fraction has been prepared from Trypanosoma brucei by sonication and differential centrifugation on sucrose gradients. This fraction is enriched 5-fold in the plasma membrane marker enzymes adenyl cyclase (EC 4.6.1.1) and ouabain-inhibitable, ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}$)-dependent adenosine triphosphatase (EC 3.6.1.3). It is also enriched up to 14-fold in iodinated surface proteins, and up to 4-fold in [³H]mannose-labeled glycoproteins, of which the major variable surface coat glycoprotein is the main constituent. Proteins of the plasma membrane fraction and other subcellular fractions have been identified by electrophoretic analysis in sodium dodecyl sulfate-polyacrylamide gradient slab gels. Several high molecular weight surface glycopeptides have been selectively investigated and partially characterized by a combination of metabolic labeling with [³H]mannose, lactoperoxidase-catalyzed surface iodination, and affinity chromatography on Con A-Sepharose. In addition to the major variable surface coat glycoprotein (estimated $\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 58 000}$), there are several minor surface glycopeptides ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 76 000, 86 000}\text{and}\text{92 000–100 000}$) which are apparent extrinsic membrane components, and two surface glycopeptides ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 42 000}\text{and}\text{130 000}$) which are intrinsic membrane components.     

Interaction of lymphocytes and macrophage cell line cells (M1 cells). I. Functional maturation and appearance of Fc receptors im M1 cells

M₁ cells, which are cell line cells established from myeloid leukemia cells of the SL strain mouse, can differentiate from blast cells ($\text{M}{}_1{}^{\mathrm{?}}$) to mature macrophages ($\text{M}{}_1{}^{\mathrm{+}}$) within 48 hr, when they are cultured with conditioned medium (CM) obtained from murine embryonic fibroblasts. While $\text{M}{}_1{}^{\mathrm{?}}$ cells have no phagocytic activity nor Fc receptor (FcR), $\text{M}{}_1{}^{\mathrm{+}}$ cells possess both characteristics. The appearance of FcR is temperature-dependent and inhibited by a metabolic inhibitor, cycloheximide. FcR on $\text{M}{}_1{}^{\mathrm{+}}$ cells is resistant to trypsin and pronase. $\text{M}{}_1{}^{\mathrm{+}}$ cells improve the viability of macrophage-depleted SL splenic lymphocytes and restore the in vitro secondary plaque forming cell response of macrophage-depleted spleen cells to particulate and soluble antigens. $\text{M}{}_1{}^{\mathrm{?}}$ cells lack this macrophage-substituting capacity. Mm₁ cells, mutant cells from M₁ cells, having FcR and higher phagocytic activity than $\text{M}{}_1{}^{\mathrm{+}}$ cells, are also devoid of this capacity.     

An adaptation of the sodium periodate/sodium borotritide labeling procedure which allows the unambiguous identification of synaptic sialoglycoproteins

An adaptation of the sodium periodate/sodium borotritide procedure for the identification of membrane sialoglycoproteins is described which eliminates interference from nonspecifically incorporated tritium. Synaptic membranes were labeled using the $\text{NaIO}{}_4\text{NaB}{}^3\text{H}{}_4$ procedure and separated by polyacrylamide gel electrophoresis. Following electrophoresis the gels were fixed, sliced, and individual slices treated with neuraminidase. Treatment with neuraminidase selectively released [³H]sialyl derivatives from the fixed glycoproteins allowing the unambiguous identification of sialoglycoproteins. The sialoglycoprotein composition of synaptic membranes and synaptic junctions was compared.     

Assembly of the vesicular stomatitis virus envelope: incorporation of viral polypeptides into the host plasma membrane

Incorporation of viral polypeptides into the host plasma membrane is an essential step in the formation of the lipoprotein envelope of vesicular stomatitis virus. A quantitative study of this process was carried out using a double-isotope labeling procedure. Infected cells were incubated for two hours with ¹⁴C-labeled amino acids, pulse-labeled with [³H]leucine and incubated for various times with an excess of non-radioactive leucine. The ${}^3\text{H}{}^{14}\text{C}$ ratio was determined for each viral polypeptide in isolated plasma membranes and in the whole cell by polyacrylamide gel electrophoresis. It was found that [³H]leucine-labeled viral polypeptides could be detected in the plasma membranes immediately following a 30-second pulse but that the ${}^3\text{H}{}^{14}\text{C}$ ratios of polypeptides in the plasma membrane did not reach the ${}^3\text{H}{}^{14}\text{C}$ ratios in the whole cells until the end of a two-minute chase period. The addition of puromycin to the cultures at the end of the pulse period did not affect subsequent incorporation of [³H]leucine-labeled polypeptides into the plasma membrane. The incorporation of various amino acid analogs into the viral polypeptides did not affect the efficiency with which they were incorporated into the plasma membranes. It is proposed that viral polypeptides are selected for incorporation into the plasma membrane from a small interior pool of completed molecules.     

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.     

Rat enterocyte Na+ transport in vitro action of parathyroid hormone and calcitonin

Parathyroid hormone (PTH) and calcitonin exert well known effects on the renal tubule which are thought to involve specific hormone receptors and adenyl cyclase. In the intestine, it is not clear whether the action of PTH and calcitonin is only indirect or also direct, and their mechanisms of action are much less well established. In the present study, possibly direct effects of PTH and calcitonin on Na⁺ transport in isolated intestinal epithelial cells of rats were investigated. In the presence of bovine PTH (1.2 I.U./ml) in the incubation medium, the Na⁺ efflux rate constant (${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$) of isolated enterocytes was significantly reduced when compared to that in control experiments with the hormone vehicle only. The mean depression of ${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$ induced by bovine PTH was 26% as compared to the control (100%) and to that induced by ouabain (4.0mM) which was 44%. No depressant effect of bovine PTH on ${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$ was observed when the isolated enterocytes were incubated with ouabain (4.0 mM). Thus, bovine PTH appeared to inhibit the ouabain-sensitive Na⁺ pump. When incubating the isolated epithelial cells in an EGTA-containing Ca²⁺-free medium, bovine PTH lost its capacity to inhibit (${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$). Thus, the presence of extracellular Ca²⁺ appeared necessary for the inhibitory effect of bovine PTH. In contrast to its effect on ${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$, bovine PTH induced no change in net Na⁺ uptake by isolated enterocytes. Moreover, no significant effect on enterocyte Na⁺ transport could be demostrated for salmon or porcine calcitonin at two different concentrations in the incubation medium. Neither bovine PTH nor salmon calcitonin induced significant changes in enterocyte cyclic AMP or cyclic GMP concentrations. It was concluded that bovine PTH, but not calcitonin, exerted a direct inhibitory effect on the ouabain-sensitive ${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$ of isolated rat enterocytes. The effect of bovine PTH occured without measurable activation of the cyclic nucleotide system but needed the presence of Ca²⁺ in the incubation medium to be operative.     

The effect of formate on cytochrome aa3 and on electron transport in the intact respiratory chain

1. Formate inhibits cytochrome $\text{c}$ oxidase activity both in intact mitochondria and submitochondrial particles, and in isolated cytochrome $\text{aa}{}_3$. The inhibition increases with decreasing pH, indicating that HCOOH may be the inhibitory species.2. Formate induces a blue shift in the absorption spectrum of oxidized cytochrome $\text{aa}{}_3\text{(a}{}^{\mathrm{3+}}\text{a}{}_3{}^{\mathrm{3+}}$) and in the half-reduced species ($\text{a}{}^{\mathrm{2+}}\text{a}{}_3{}^{\mathrm{3+}}$). Comparison with cyanide-induced spectral shifts, towards the red, indicates that formate and cyanide have opposite effects on the $\text{aa}{}_3$ spectrum, both in the fully oxidized and the half-reduced states. The formate spectra provide a new method of obtaining the difference spectrum of $\text{a}{}_3{}^{\mathrm{2+}}$ minus $\text{a}{}_3{}^{\mathrm{3+}}$, free of the difficulties with cyanide (which induces marked high → low spin spectral shifts in cytochrome $\text{a}{}_3{}^{\mathrm{3+}}$) and azide (which induces peak shifts of cytochrome $\text{a}{}^{\mathrm{2+}}$ towards the blue in both α- and Soret regions).3. The rate of formate dissociation from cytochrome $\text{a}{}^{\mathrm{2+}}\text{a}{}_3{}^{\mathrm{3+}}\text{-}\text{HCOOH}$ is faster than its rate of dissociation from $\text{a}{}^{\mathrm{3+}}\text{a}{}_3{}^{\mathrm{3+}}\text{-}\text{HCOOH}$, especially in the presence of cytochrome $\text{c}$. The $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ for formate inhibition of respiration is a function of the reduction state of the system, varying from 30 mM (100% reduction) to 1 mM (100% oxidation) at pH 7.4, 30 °C.4. Succinate-cytochrome $\text{c}$ reductase activity is also inhibited by formate, in a reaction competitive with succinate and dependent on [formate]².5. Formate inhibition of ascorbate plus $\text{N,N,N′,N′-}\text{tetramethyl}\text{-p-}\text{phenyl-enediamine}$ oxidation by intact rat liver mitochondria is partially released by uncoupler addition. Formate is permeable through the inner mitochondrial membrane and no differences in ‘on’ or ‘off’ inhibition rates were observed when intact mitochondria were compared with submitochondrial particles.6. NADH-cytochrome $\text{c}$ reductase activity is unaffected by formate in submitochondrial particles, but mitochondrial oxidation of glutamate plus malate is subject both to terminal inhibition at the cytochrome $\text{aa}{}_3$ level and to a slow extra inhibition by formate following uncoupler addition, indicating a third site of formate action in the intact mitochondrion.     

Lymphocyte receptors for polyclonal T mitogens: II. High-molecular-weight glycoproteins are the best candidate sites for mitogenic action by concanavalin A and galactose oxidase

Murine lymphocytes oxidized by galactose oxidase were radiolabeled by reduction with NaB³H₄. The labeled cells were incubated with Con A and the Con A-Con A receptor complexes formed in situ on the viable cells were isolated by immuno-precipitation with anti-Con A serum and fixed Staphylococcus aureus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography analysis of the precipitates demonstrated four high-molecular-weight glycoproteins which were oxidized by GO and which bound Con A. These same four glycoproteins were also oxidized and labeled by $\text{IO}{}_4\text{NaB}{}^3\text{H}{}_4$. [³H]Tyrosine biosynthetic labeling identified these four plus several other Con A receptors. Because Con A sterically inhibits GO mitogenic stimulation, these four glycoproteins are likely to represent the necessary sites of oxidative mitogenic action and are good candidates for the targets of Con A mitogenesis.     

An enzymatic method for [32P]phosphoenolpyruvate synthesis

A convenient method for the enzymatic synthesis of [³²P]phosphoenolpyruvate from [γ-³²P]ATP using partially pufified phosphoenolpyruvate carboxykinase from Escherichia coli is described. The synthesis was shown to convert essentially all the [γ-³²P]ATP to [³²P]phosphoenolpyruvate, which was subsequently separated from residual [γ-³²P]ATP and $\text{[}{}^{32}\text{P}\text{]P}{}_{\mathrm{<mtext>i</mtext>}}$ by chromatography on AG-1-X8-bicarbonate resin.