Cell-free labeling in thyroid rough microsomes of lipid-linked and protein-linked oligosaccharides: II. Glucosylated units

Pig thyroid rough microsomes catalyzed the transfer of glucose from UDP-[¹⁴C]Glc to glycolipids extractable with chloroform/methanol, glycolipids extractable with a water-saturated chloroform/methanol and to a residual material. Kinetics of labeling were compatible with a precursor-product relationship between the second type of glycolipid and residuals.The [¹⁴C] Glc-glycolipids soluble in CHCl₃/CH₃OH/H₂O, 10 : 10 : 3, behaved on DEAE-cellulose mainly as pyrophospho derivatives, with some less acidic radioactivity, probably $\text{dolichol}\text{-P-[}{}^{14}\text{C}\text{]}\text{Glc}$. Their saccharide moieties released by mild acid appeared polydisperse on paper chromatography, a part of them being estimated larger than a nonasaccharide marker GlcNAc-[Man]₈. The ¹⁴C-labeled glucosylated glycoproteins have represented all the considerable polymeric label remaining after lipid extraction. Their pronase glycopeptides were submitted to a differential reductive alkaline hydrolysis and it was concluded that their [¹⁴C] glucose belongs mainly to N-glycosically linked units. On gel filtration, the released saccharides exhibited an average size of nine monosaccharide units (from six to twelve with a relatively high proportion of material containing more than nine sugars).In a [¹⁴C] Glc-microsomal extract, 29% of the non-lipid radioactivity was found immunoreactive with an antiserum to pig thyroglobulin.     

Transfer of N, N'-diacetylchitobiose from dolichyl diphosphate into a gray matter membrane glycoprotein

Gray matter and white matter membranes catalyze the transfer of label from UDP-N-acetyl-[${}^{14}\text{C}$] glucosamine into N-acetyl[${}^{14}\text{C}$]glucosaminyl-pyrophosphoryl-dolichol, N,N′-diacetyl [${}^{14}\text{C}$]chitobiosyl-pyrophosphoryl-dolichol, and N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein. Gel filtration of the Pronase digests of gray matter N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein reveals two N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide fractions. One fraction (A) contains approximately eight glycose units. All of the radioactivity is at nonreducing termini and can be released by treatment with an exo-β-N-acetylglucosaminidase. A smaller N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide (B) is recovered in the elution volume expected for an asparaginyl disaccharide. Structural studies show that the labeled saccharide unit in glycopeptide B is N,N′-diacetyl[${}^{14}\text{C}$]chitobiose. The linkage between the ${}^{14}\text{C}$-labeled disaccharide and the polypeptide has the properties of an N-glycosidic attachment to asparagine. Only the larger N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide (A) is found in Pronase digests of white matter membrane N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein after incubation with UDP-N-acetyl[${}^{14}\text{C}$]glucosamine. When gray matter membranes are incubated with UDP-N-acetyl[${}^{14}\text{C}$]glucosamine in the presence of tunicamycin or UMP, the labeling of glycolipid and the asparaginyl disaccharide is inhibited. UMP and tunicamycin have no effect on the transfer of N-acetyl[${}^{14}\text{C}$]glucosamine to external acceptor sites of the larger glycopeptide (A). The transfer of N,N′-diacetyl[${}^{14}\text{C}$]-chitobiose from carrier lipid to protein is observed when extensively washed membranes containing endogenous, prelabeled ${}^{14}\text{C}$-labeled glycolipids are incubated in the presence or absence of unlabeled GDP-mannose. UMP treatment of the prelabeled membranes selectively discharged over 80% of the label from N-acetyl[${}^{14}\text{C}$]glucosaminyl-pyrophosphoryl-dolichol, but had no effect on the transfer of the ${}^{14}\text{C}$-labeled disaccharide to protein. All of these results are concordant with transfer of N,N′-diacetylchitobiose from dolichyl diphosphate to gray matter glycoprotein. The major membrane glycoprotein labeled by the lipid-mediated [${}^{14}\text{C}$]disaccharide transfer reaction has an apparent molecular weight of 24,000. Tunicamycin prevents the enzymatic labeling of the gray matter glycoprotein having an apparent molecular weight of 24,000.     

Modification of sialic acid metabolism of murine erythroleukemia cells by analogs of N-acetylmannosamine

Two analogs of N-acetylmannosamine, $\text{2-}\text{acetamido}\text{-1,3,4,6-}\text{tetra}\text{-O-}\text{acetyl}\text{-2-}\text{deoxy-}\text{d}\text{-mannopyranose}$ (Ac₄-NAcMan) and the 2-trifluoroacetamido derivative (Ac₄F₃-NAcMan), were synthesized as potential inhibitors of the formation of sialic acid-containing glycoconjugates and were examined for their ability to modify the incorporation of $\text{N-[}{}^3\text{H]acetylmannosamine}$ into cellular glycoconjugates of Friend murine erythroleukemia cells. Ac₄F₃-NAcMan and Ac₄-NAcMan inhibited cellular replication in suspension culture at concentrations of 0.02 and 0.08 mM, respectively. The cytotoxicity of Ac₄-NAcMan was relatively reversible, whereas that produced by Ac₄F₃-NAcMan was not, as judged by measurement of the cloning efficiencies of cells exposed to these agents. The analogs inhibited incorporation of $\text{N-[}{}^3\text{H]acetylmannosamine}$ into ethanol-soluble and -insoluble materials. Separation of ethanol-soluble metabolites by HPLC demonstrated that Ac₄F₃-NAcMan caused accumulation of radioactivity from $\text{N-[}{}^3\text{H]acetylmannosamine}$ in CMP-N-acetylneuraminic acid (CMP-NeuNAc) equal to the decrease in macromolecular-bound ³H caused by this agent. In contrast, similar exposure to Ac₄-NAcMan produced a large increase in the amount of radioactivity in ethanol-soluble N-acetylneuraminic acid while decreasing the amount of label from $\text{N-[}{}^3\text{H]acetylmannosamine}$ in cellular CMP-NeuNAc, suggesting that the analogs differ in their biochemical sites of action. Treatment of cells with either analog increased the amount of neuraminidase-hydrolyzable sialic acid-like material on the cell surface; this appeared to be due to the incorporation of the analogs into cellular glycoconjugates, since incubation of cells with ³H-labeled analogs resulted in the appearance of radioactivity in cellular ethanol-insoluble and neuraminidase-hydrolyzable material. Incubation of cells with Ac₄-NAcMan labeled with ¹⁴C in the 4-O-acetyl group further demonstrated that incorporation occurred with approx. 50% retention of this substituent. Thus, both the amount and the nature of the surface sialic acid constituents of treated cells were altered, suggesting that these or similar analogs could potentially be used to modify cellular membrane function.     

Studies on the metabolism of guanidine compounds in mammals

[¹⁴C]Guanidine was observed in the urine after subcutaneous administration to rats of l-[guanidino-¹⁴C]arginine or l-[guanidino-¹⁴C]canavanine. [${}^{14}\text{C}$]Hydroxyguanidine was additionally detected in the urine after injection of dl-[guanidino-¹⁴C]canavanine. These ${}^{14}\text{C}$ metabolites were characterized by high-voltage electrophoresis and paper chromatography, by enzymatic conversion of [¹⁴C]hydroxyguanidine to [¹⁴C]guanidine, and by repeated recrystallization of isolated urinary [${}^{14}\text{C}$]guanidine as the picrate salt with no significant loss of specific activity. These experiments demonstrate that both l-arginine and l-canavanine can serve as precursors of guanidine in the rat.     

N(4)-amino-and N(4)-hydroxycytosines as base analogue mutagens

N(4)-Aminocytosine [N(4)NH₂C] and N(4)-amino-2′-deoxycytidine [N(4)NH₂dC] are highly mutagenic for Escherichia coli and phage φ 80 but not for T₄. There is some evidence that they are incorporated into the φ 80 DNA but $\text{[}{}^{14}\text{C}\text{]-N(4)}\text{NH}{}_2\text{C}$ could not be detected in the bacterial DNA.N(4)-Hydroxy-5,6-dihydro-6-hydroxylaminodeoxycytidine (di-NHOH-dC) is mutagenic for φ 80 and E. coli, but N(4)-hydroxydeoxycytidine [N(4)OH-dC] only has a strong inactivating effect.     

Respiration-driven proton translocation with nitrite and nitrous oxide in Paracoccus denitrificans

(1) $\text{H}$⁺/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O₂, NO^?₂ or N₂O. Under optimal H⁺-translocation conditions, the ratios $\text{H}{}^{\mathrm{+}}\text{O}$, $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as exogenous substrate, addition of NO^?₂ or N₂O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were ?0.84, ?2.33 and ?1.90, respectively. (3) The $\text{H}{}^{\mathrm{+}}\text{oxidant}$ ratios, mentioned in item 2, were not altered in the presence of $\text{valinomycin}\text{K}{}^{\mathrm{+}}$ and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O₂, NO^?₂ and N₂O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO^?₂, N₂O or O₂ pass two H⁺-translocating sites. The $\text{H}{}^{\mathrm{+}}\text{electron}$ acceptor ratios predicted by this scheme are in good agreement with the experimental values.     

Presteady-state kinetic study of the elementary processes in the chymotrypsin-catalyzed hydrolysis of specific ester substrate. Rate-limiting association process due to the secondary binding.

Presteady-state kinetic studies of α-chymotrypsin-catalyzed hydrolysis of a specific chromophoric substrate, N-(2-furyl)acryloyl-l-tryptophan methyl ester, were performed by using a stopped-flow apparatus both under [E]₀ ? [S]₀ and [S]₀ ? [E]₀ conditions in the pH range of 5–9, at 25 °C. The results were accounted for in terms of the three-step mechanism involving enzyme-substrate complex (E · S) and acylated enzyme (ES′); no other intermediate was observed. This substrate was shown to react very efficiently, i.e., the maximum of the second-order acylation rate constant ($\text{k}{}_2\text{K}{}_{\mathrm{s}}\text{′}\text{)}{}_{\mathrm{<mtext>max</mtext>}}\text{= 4.2 × 10}{}^7\text{M}{}^{\mathrm{?1}}\text{s}{}^{\mathrm{?1}}$. The limiting values of K_s′ (dissociation constant of E · S), K₂ (acylation rate) and k₃ (deacylation rate) were obtained from the pH profiles of these parameters to be 0.6 ± 0.2 × 10^?5 m, 360 ± 15 s^?1 and 29.3 ± 0.8 s^?1, respectively. Likewise small values were observed for K_i of N-(2-furyl)-acryloyl-l-tryptophan and N-(2-furyl)acryloyl-d-tryptophan methyl ester and K_m of N-(2-furyl)acryloyl-l-tryptophan amide. The strong affinities observed may be due to intense interaction of β-(2-furyl)acryloyl group with a secondary binding site of the enzyme. This interaction led to a $\text{k}{}_{\mathrm{?1}}\text{k}{}_2$ value lower than unity, i.e., the rate-limiting process of the acylation was the association, even with the relatively low k₂ value of this methyl ester substrate, compared to those proposed for labile p-nitrophenyl esters.     

Aromatic origin of cyclopentenoid metabolites

Oxidative cleavage of aromatic compounds is often part of a degradative process and is widely observed in nature. The immediate catabolic products can sometimes cyclize or rearrange to new secondary metabolites. The enzymatic contraction of a dehydroisocoumarin to yield cyclopentenoid metabolites in Cryptosporiopsis sp. is reported. The label distribution of (+) cryptosporiopsin, a chlorinated cyclopentenone, was determined by analysis of the [¹³C]nmr of [1-¹³C] and [2-¹³C]acetate enriched-cryptosporiopsin. The putative aromatic precursor of cyclopentenoid metabolites, 2,3-dihydro-6,8-dihydroxy-2-methylisocoumarin (6), was isolated from Aspergillus terreus. This metabolite (6) was prepared doubly labeled ($\text{T}{}^{14}\text{C}$). The aromatic origin of the Cryptosporiopsis chlorinated cyclopentenoid metabolites was rigorously proven from feeding experiments with doubly labeled compound 6. A related but nonchlorinated metabolite, terrein, was isolated from A. terreus and was also shown to be derived from [$\text{T}{}^{14}\text{C}\text{]-2,3-dihydro-6,8-dihydroxy-2-methylisocoumarin}$.     

Equilibrium constants under physiological conditions for the reactions of L-phosphoserine phosphatase and pyrophosphate: L-serine phosphotransferase

The observed equilibrium constants (K_obs) for the l-phosphoserine phosphatase reaction [EC 3.1.3.3] have been determined under physiological conditions of temperature (38 °C) and ionic strength (0.25 m) and physiological ranges of pH and free [Mg²⁺]. Using Σ and square brackets to indicate total concentrations $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{Σ L-serine][Σ P}{}_{\mathrm{i}}\text{]}\text{Σ L-phosphoserine]H}{}_2\text{O]}\text{, K =}\text{L-H · serine}{}^{\mathrm{\pm }}\text{]HPO}{}_4{}^{\mathrm{2?}}\text{]}\text{[L-H · phosphoserine}{}^{\mathrm{2?}}\text{]H}{}_2\text{O]}$. The value of K_obs has been found to be relatively sensitive to pH. At 38 °C, K⁺] = 0.2 m and free [Mg²⁺] = 0; K_obs = 80.6 m at pH 6.5, 52.7 m at pH 7.0 [ΔG_obs⁰ = ?10.2 kJ/mol (?2.45 kcal/mol)], and 44.0 m at pH 8.0 ([H₂O] = 1). The effect of the free [Mg²⁺] on K_obs was relatively slight; at pH 7.0 ([K⁺] = 0.2 m) K_obs = 52.0 m at free [Mg²⁺] = 10^?3, m and 47.8 m at free [Mg²⁺] = 10^?2, m. K_obs was insignificantly affected by variations in ionic strength (0.12–1.0 m) or temperature (4–43 °C) at pH 7.0. The value of K at 38 °C and I = 0.25 m has been calculated to be 34.2 ± 0.5 m [ΔG_obs⁰ = ?9.12 kJ/mol (?2.18 kcal/ mol)]([H₂O] = 1). The K for the phosphoserine phosphatase reaction has been combined with the K for the reaction of inorganic pyrophosphatase [EC 3.6.1.1] previously estimated under the same physiological conditions to calculate a value of 2.04 × 10⁴, m [ΔG_obs⁰ = ?28.0 kJ/mol (?6.69 kcal/mol)] for the K of the pyrophosphate:l-serine phosphotransferase [EC 2.7.1.80] reaction. $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{[}\text{Σ L-serine}\text{][}\text{Σ}\text{P}{}_{\mathrm{<mtext>i</mtext>}}\text{]}\text{[}\text{Σ L-phosphoserine}\text{][}\text{H}{}_2\text{O}\text{]}\text{, K =}\text{[L-H · serine}{}^{\mathrm{\pm }}\text{]HPO}{}_4{}^{\mathrm{2?}}\text{]}\text{[L-H · phosphoserine}{}^{\mathrm{2?}}\text{]H}{}_2\text{O}$. Values of K_obs for this reaction at 38 °C, pH 7.0, and I = 0.25 m are very sensitive to the free [Mg²⁺], being calculated to be 668 [ΔG_obs⁰ = ?16.8 kJ/mol (?4.02 kcal/mol)] at free [Mg²⁺] = 0; 111 [ΔG_obs⁰ = ?12.2 kJ/mol (?2.91 kcal/mol)] at free [Mg²⁺] = 10^?3, m; and 9.1 [ΔG_obs⁰ = ?5.7 kJ/mol (?1.4 kcal/mol) at free [Mg²⁺] = 10^?2, m). K_obs for this reaction is also sensitive to pH. At pH 8.0 the corresponding values of K_obs are 4000 [ΔG_obs⁰ = ?21.4 kJ/mol (?5.12 kcal/mol)] at free [Mg²⁺] = 0; and 97.4 [ΔG_obs⁰ = ?11.8 kJ/ mol (?2.83 kcal/mol)] at free [Mg²⁺] = 10^?3, m. Combining K_obs for the l-phosphoserine phosphatase reaction with K_obs for the reactions of d-3-phosphoglycerate dehydrogenase [EC 1.1.1.95] and l-phosphoserine aminotransferase [EC 2.6.1.52] previously determined under the same physiological conditions has allowed the calculation of K_obs for the overall biosynthesis of l-serine from d-3-phosphoglycerate. $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{[}\text{Σ L-serine}\text{][}\text{Σ NADH}\text{][}\text{Σ}\text{P}{}_{\mathrm{<mtext>i</mtext>}}\text{][}\text{Σ}\text{α-}\text{ketoglutarate}\text{]}\text{[Σ d-3-}\text{phosphoglycerate}\text{][Σ NAD}{}^{\mathrm{+}}\text{][Σ L-glutamat0]}$ The value of K_obs for these combined reactions at 38 °C, pH 7.0, and I = 0.25 m (K⁺ as the monovalent cation) is 1.34 × 10^?2, m at free [Mg²⁺] = 0 and 1.27 × 10^?2, m at free [Mg²⁺] = 10^?3, m.     

Tissue-specificity of N-nitrosodibutylamine metabolism in Sprague-Dawley rats

The distribution and metabolism of $\text{N-[}{}^{14}\text{C}\text{]}$nitrosodibutylamine were studied in Sprague-Dawley rats. The results indicated that in addition to the liver, metabolism of the substance occurred in the nasal mucosa, the lung and the oesophagus. Metyrapone and diethyldithiocarbamate reduced the production of ¹⁴CO₂ from $\text{N-[}{}^{14}\text{C}\text{]}$nitrosodibutylamine by all these tissues. There was no indication of metabolic capacity in the urinary bladder or the kidney. The results fit with the assumption that tumours of the urinary tract are induced by metabolites reaching these tissues via the urine. Besides the liver, the oesophagus and the lung are target tissues for the carcinogenicity of N-nitrosodibutylamine in Sprague-Dawley rats and in these tissues the local formation of reactive metabolites may play a role in the pathogenesis of N-nitrosodibutylamine-induced lesions.     

Assay of Δ1-piperideine-2-carboxylate and synthesis of l-[14C]pipecolate from dl-[14C]pipecolate

Four assay methods were tested for the measurement of Δ¹-piperideine-2-carboxylate, a proposed alicyclic ketimino acid intermediate in the pathway of lysine metabolism to l-pipecolate, and the product of d-amino acid oxidase on d-pipecolate. The method using Δ¹-piperideine-2-carboxylate reductase from Pseudomonas putida was found to be most sensitive and specific. Measurement of Δ¹-piperideine-2-carboxylate by reduction with NaBH₄ and ninhydrin assay of the resultant pipecolate, by direct acidic ninhydrin assay, and by o-aminobenz-aldehyde assay were less desirable because of lower sensitivity and specificity. Two synthetic methods for preparing l-[¹⁴C]pipecolate from the racemic dl-[¹⁴C]pipecolate were investigated. Incubation of dl-[¹⁴C]pipecolate with a combination of d-amino acid oxidase and Δ¹-piperideine-2-carboxylate reductase or d-amino acid oxidase and NaBH₄ totally inverted the d-isomer to the l-isomer, with $\text{Δ}{}^1\text{-[}{}^{14}\text{C]piperideine-2-carboxylate}$ as an intermediate in each cycle of interconversion. No purification except desalting through a Dowex 50 (H⁺) column was necessary in order to recover l-[¹⁴C]pipecolate in pure form. The yield was 95–97% compared to <50% in the conventional method.     

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.     

An overall radioassay for the first three reactions of de novo pyrimidine biosynthesis

A sensitive radioassay is described for the overall biosynthetic activity of the multienzymatic protein which catalyzes the first three reactions of de novo pyrimidine biosynthesis in mammals. The ability of the multienzymatic protein to synthesize dihydroorotate can be assayed using $\text{[}{}^{14}\text{C]HCO}{}_3{}^{\mathrm{?}}$, l-[¹⁴C]aspartate, or [${}^{14}\text{C}$] carbamyl phosphate as substrate. The synthesis of the final product, l-dihydroorotate, may be coupled to synthesis of orotidine 5′-monophosphate to overcome the unfavorable equilibrium existing between l-dihydroorotate and its precursor, N-carbamyl-l-aspartate, in the physiological pH range (Christopherson, R. I., and Jones, M. E., 1979, J. Biol. Chem.254, 12506–12512). l-Aspartate and all pyrimidine intermediates from carbamyl phosphate to orotidine 5′-monophosphate can be clearly separated by ion-exchange chromatography in a single dimension on polyethyleneimine-cellulose chromatograms and carbamyl phosphate and its degradation product cyanate may be quantitated directly along with the other intermediates.     

Effect of N-alkyl-N,N,N-trimethylammonium ions on phosphatidylcholine model membrane structure as studied by 31P-NMR

The interaction of |C_nH_2n+1N⁺(CH₃)₃| · I^? (n = 3, 6, 9, 12, 14, 16 or 18) with egg-yolk phosphatidylcholine-water dispersions has been studied by ³¹P-NMR spectroscopy. It is shown that the effective anisotropy of ³¹P chemical shift (?Δσ_eff) of the lamellar phospholipid liquid-crystalline phase L_α increases with increasing concentration and alkyl chain length of the drug. Addition of $\text{|}\text{C}{}_6\text{H}{}_{13}\text{N}{}^{\mathrm{+}}\text{(CH}{}_3\text{)}{}_3\text{| ·I}{}^{\mathrm{?}}$ or |C₉H₁₉N⁺(CH₃)₃|·I^? to the phospholipid-water dispersion at a molar ratio ammonium salt:phospholipid > 0.8 induces in the dispersion a structure with an effective isotropic phospholipid motion. This structure is unstable and slowly transforms into the hexagonal phase. These effects have not been observed in phospholipid-water dispersions mixed with the ammonium derivatives with the longer alkyl chains n  12, 14, 16 or 18. It is proposed that these results might explain the effects of the investigated drugs on the nerve, muscle and bacterial cells.     

Effects of N,N′-dicyclohexylcarbodiimide on isolated and reconstituted cytochrome b-c1 complex from bovine heart mitochondria

N,N′-Dicyclohexylcarbodiimide (DCCD) inhibits the activity of ubiquinol-cytochrome c reductase in the isolated and reconstitued mitochondrial cytochrome b-c₁ complex. DCCD inhibits equally electron flow and proton translocation (i.e., the $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ ratio is not affected) catalysed by the enzyme reconstituted into phospholipid vesicles. The inhibitory effects are accompanied by structural alterations in the polypeptide pattern of both isolated and reconstituted enzyme. Cross-linking was observed between subunits V (iron-sulfur protein) and VII, indicating that these polypeptides are in close proximity. A clear correlation was found between the kinetics of inhibition of enzymic activity and the cross-linking, suggesting that the two phenomena may be coupled. Binding of [¹⁴C]DCCD was also observed, to all subunits with the isolated enzyme and preferentially to cytochrome b with the reconstituted vesicles; in both cases, however, it was not correlated kinetically with the inhibition of the enzymic activity.     

Structural studies of the M blood group determinant

Structural studies of homozygous glycophorin A^M were undertaken by monitoring the ¹³C methyl resonances of ¹³C reductively methylated glycophorin A^M (contains five $\text{N}{}^{\mathrm{?}}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ Lys residues, and the N-terminal $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C]}\text{dimethyl}$ Ser residues) in various forms of glycosylation. The results indicate that removal of the α-d-NeuAc residues does not affect the structure about the N-terminal Ser residue. However, removal of the fifteen O-linked oligosaccharide units results in a structural effect about the N-terminal Ser residue. Partial methylation experiments performed on native glycophorin A^M and deglycosylated glycophorin A^M indicate that methylation of the lysine residue(s) may influence the structure about the N-terminal Ser residue, especially in the case of deglycosylated A^M.     

The alkaline hydrolysis of phosphotriesters in alkylated mammalian DNA

Isolated DNA was alkylated with $\text{N-[}{}^{14}\text{C]methyl}\text{-N-}\text{nitrosourea}$ or $\text{N-[}{}^{14}\text{C]ethyl}\text{-N-}\text{nitrosourea}$. Sedimentation analysis of the alkylated DNA before and after alkaline hydrolysis was used to determine the number of single-strand breaks introduced by hydrolysis of the triesters. Vacuum distillation from alkylated DNA solutions before and after alkaline hydrolysis was used to determine the numbers of triesters hydrolysing to the alcohol.     

Invivo conversion of 3-ketoceramide to ceramide in rat liver

A doubly labeled 3-ketoceramide, [1-¹⁴C] lignoceroyl [1-³H₂] 3-ketosphingosine ($\text{3H}{}^{14}\text{C}$ ratio, 3.61) was injected into the left ventricle of rat heart. The ceramide isolated from the livers of the animals after 1 hr incubation contained an equal $\text{3H}\text{>}{}^{14}\text{C}$ ratio of 3.60. This finding strongly supports the existence for direct conversion of 3-ketoceramide to ceramide in rat liver.     

Mevalonate metabolism: Effect of K2Cr2O7, a renal tubular toxin

K₂Cr₂O₇ was given to one member of 10 pairs of rats 2.5 hours before injection of $\text{RS}\text{-[5-}{}^{14}\text{C]}$mevalonate. The poisoned rats expired 30.8 ± 3.5% less ¹⁴CO₂ than their unpoisoned controls in the 2.5 hours after mevalonate injection (p < .001) and incorporated 16.3 ± 7.1% less label into renal (p<.05) and 63.7 ± 12.0% more label into hepatic unsaponifiable lipids (p < .001). K₂Cr₂O₇ had no effect on the oxidation of Na[1-¹⁴C]octanoate. These changes occurred at least 14 hours before any previously demonstrated effect of K₂Cr₂O₇ on the kidney and represent an early biochemical expression of renal tubular damage.     

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.