Quantitative importance of non-skeletal-muscle sources of N tau-methylhistidine in urine.

Direct measurement of N tau-methylhistidine turnover in skeletal muscle, skin and gastrointestinal muscle indicates that these three tissues contribute only 24.9, 6.8 and 9.8% of the total urinary excretion. Measurement of the decay rate of radioactively labelled N tau-methylhistidine in urine indicates that skeletal muscle accounts for 74.5% of the urinary excretion and this is probably an overestimate. These results suggest that the common assumption, that N tau-methylhistidine in urine originates almost entirely from skeletal muscle, may be wrong.     

An analysis of errors in estimation of the rate of protein synthesis by constant infusion of a labelled amino acid.

Rates of protein synthesis in tissues can be calculated from the specific radioactivity of free and protein-bound amino acids at the end of a constant infusion of a labelled amino acid (Garlick, Millward & James (1973) Biochem. J. 136, 935--945]. The simplifying assumptions used in these calculations have been criticized [Madsen, Everett, Sparrow & Fowkes (1977) FEBS Lett. 79, 313--316]. A more detailed analysis using a programmable desk-top calculator is described, which shows that the errors introduced by the simplifying assumptions are small, particularly when the specific radioactivity of the free amino acid rises rapidly to a constant value.     

Importance of experimental conditions in evaluating the malonyl-CoA sensitivity of liver carnitine acyltransferase. Studies with fed and starved rats.

The experiments reconfirm the powerful inhibitory effect of malonyl-CoA on carnitine acyltransferase I and fatty acid oxidation in rat liver mitochondria (Ki 1.5 microM). Sensitivity decreased with starvation (Ki after 18 h starvation 3.0 microM, and after 42 h 5.0 microM). Observations by Cook, Otto & Cornell [Biochem. J. (1980) 192, 955--958] and Ontko & Johns [Biochem. J. (1980) 192, 959--962] have cast doubt on the physiological role of malonyl-CoA in the regulation of hepatic fatty acid oxidation and ketogenesis. The high Ki values obtained in the cited studies are shown to be due to incubation conditions that cause substrate depletion, destruction of malonyl-CoA or generation of excessively high concentrations of unbound acyl-CoA (which offsets the competitive inhibition of malonyl-CoA towards carnitine acyltransferase I). The present results are entirely consistent with the postulated role of malonyl-CoA as the primary regulatory of fatty acid synthesis and oxidation in rat liver.     

Quantitative determination of urinary N-tau-methylhistidine output as an index of myofibrillar protein degradation

N tau-Methylhistidine(3-methylhistidine) in urine of the rat is mainly derived from the degradation of actin and myosin in skeletal muscle, intestine and skin. The fractional degradation rates of the myosin-actin pools of these tissues were calculated from the time course of increase in the specific radioactivities of N tau-methylhistidine after daily administration of [methyl-14C]methionine to young adult rats under conditions of restricted food intake. The contributions to urinary excretion of N tau-methylhistidine from the three tissues were calculated from the fractional degradation rates and N tau-methylhistidine contents of the three tissues; 75.6% for skeletal muscle, 2.2% for intestine and 22.2% for skin. The results show that the skeletal muscle is the major source of urinary N-tau-methylhistidine output, but the contribution of skin is not negligible in rats. The specific radioactivity of N tau-methylhistidine in urine was much higher than that of skeletal muscle. The fractional degradation rates of myosin and actin in skeletal muscle had similar values. Although the specific radioactivities of N tau-methylhistidine in myosin and actin were very different, the mean value was similar to that in mixed skeletal muscle.     

Active sites of beta-lactamases. The chromosomal beta-lactamases of Pseudomonas aeruginosa and Escherichia coli.

An acyl-enzyme was isolated from certain chromosomal beta-lactamases and a penicillin. The penicillin was cloxacillin which, although it is a substrate for these enzymes, has such a low kcat. that it functions as an inhibitor. The enzymes were from the mutant of Pseudomonas aeruginosa 18 S that produces the beta-lactamase constitutively [Flett, Curtis & Richmond (1976) J. Bacteriol. 127, 1585-1586; Berks, Redhead & Abraham (1982) J. Gen. Microbiol., in the press] and from Escherichia coli K-12 (the ampC beta-lactamase) [Boman, Nordström & Normak (1974) Ann. N.Y. Acad. Sci. 235, 569-586]. The acyl-enzymes have been degraded to determine the residue labelled, and the sequence around it. The residue labelled is serine. The sequences around the labelled serine in these two beta-lactamases are exceedingly similar. However, the sequences are quite different from those around the active site serine in the beta-lactamases previously studied. There is thus more than one class of serine beta-lactamases.     

The exocellular beta-lactamase of Streptomyces albus G. Purification, properties and comparison with the exocellular DD-carboxypeptidase.

The exocellular beta-lactamase of Streptomyces albus G has been purified to near protein homogeneity. It consists of one single polypeptide chain of mol.wt. 30 000-31 000, has a rather low isoelectric point (at pH 6.0) and contains less lysine (2.1%) and more half-cystine residues than most beta-lactamases from other Gram-positive bacteria. Penicillins are much better substrates than delta 3-cephalosporins; the catalytic-centre activity of good penicillin substrates is 333-500 s-1. The exocellular, mol.wt. 17 000 DD-carboxypeptidase of S. albus G [previously purified to protein homogeneity; Duez, Frère, Geurts, Ghuysen, Dierickx & Delcambe (1978) Biochem. J. 175, 793-800] behaves as an exceedingly poor beta-lactamase, hydrolysing benzylpenicillin into benzylpenicilloate 5 x 10(-6)-fold less rapidly than does the exocellular beta-lactamase. To all appearances, the beta-lactamase has no bivalent cation requirement whereas, as shown elsewhere [Dideberg, Charlier, Dupont, Vermeire, Frère & Ghuysen (1980) FEBS Lett. 117, 212-214, and Dideberg, Joris, Frère, Ghuysen, Weber, Robaye, Delbrouck & Roelands (1980) FEBS Lett. 117, 215-218], the DD-carboxypeptidase possesses one essential Zn2+ ion per molecule. Peptide 'mapping' and immunological studies suggest that the two Streptomyces enzymes probably have very different structural and mechanistic properties.     

Effect of a protein-free diet on muscle protein turnover and nitrogen conservation in euthyroid and hyperthyroid rats.

Although protein turnover in skeletal muscle is increased in hyperthyroidism and decreased in hypothyroidism, a deficient protein intake tends to increase serum T3 (tri-iodothyronine) while decreasing muscle protein turnover. To determine whether this diet-induced decrease in protein turnover can occur independent of thyroid status, we have examined muscle protein turnover and nitrogen conservation in hyperthyroid rats fed on a protein-free diet. After inducing hyperthyroidism by giving 20 micrograms of T3/100g body wt. daily for 7 days, groups of euthyroid and hyperthyroid animals were divided into subgroups fed on basal and protein-free diets. Muscle protein turnover was measured by N tau-methylhistidine excretion and [14C]tyrosine infusion. Urinary nitrogen output of euthyroid and hyperthyroid animals fed on the protein-free diet was also measured. Although hyperthyroidism increased the baseline rates of muscle protein synthesis and degradation, it did not prevent a decrease in these values in response to protein depletion. Furthermore, hyperthyroid rats showed greatly decreased nitrogen excretion in response to the protein-free diet, although not to values for euthyroid rats. These findings suggest that protein depletion made the experimental animals less responsive to the protein-catabolic effects of T3.     

Stimulation of the alternative oxidase of Neurospora crassa by Nucleoside phosphates.

The alternative-oxidase-mediated succinate oxidase activity of Neurospora crassa decreases drastically when mitochondria are fractionated into submitochondrial particles or treated with deoxycholate. The activity, however, can be completely restored in the presence of nucleoside 5'-monophosphates. The purine nucleoside 5'-monophosphates are more effective than the pyrimidine homologues. 5'-GMP gives a 10-fold stimulation of the alternative-oxidase-mediated succinate oxidase activity in submitochondrial particles. A comparison is made with the results obtained earlier with Moniliella tomentosa [Hanssens & Verachtert (1976) J. Bacteriol. 125, 825--835; Vanderleyden, Van Den Eynde & Verachtert (1980) Biochem. J. 186, 309--316].     

A synopsis of the tribe Lachnophorini,with a new genus of Neotropical distribution and a revision of the Neotropical genus Asklepia Liebke, 1938 (Insecta,Coleoptera, Carabidae)

This synopsis provides an identification key to the genera of Tribe Lachnophorini of the Western and Eastern Hemispheres including five genera previously misplaced in carabid classifications. The genus Asklepia Liebke, 1938 is revised with 23 new species added and four species reassigned from Eucaerus LeConte, 1853 to Asklepia Liebke, 1938. In addition, a new genus is added herein to the Tribe: Peruphorticus gen. n. with its type species P. gulliveri sp. n. from Perú. Five taxa previously assigned to other tribes have adult attributes that make them candidates for classification in the Lachnophorini: Homethes Newman, Aeolodermus Andrewes, Stenocheila Laporte de Castelnau, Diplacanthogaster Liebke, and Selina Motschulsky are now considered to belong to the Lachnophorini as genera incertae sedis. Three higher level groups are proposed to contain the 18 recognized genera: the Lachnophorina, Eucaerina, and incertae sedis.Twenty-three new species of the genus Asklepia are described and four new combinations are presented. They are listed with their type localities as follows: (geminata species group) Asklepia geminata (Bates, 1871), comb. n, Santarém, Rio Tapajós, Brazil; (hilaris species group) Asklepia campbellorum Zamorano & Erwin, sp. n., 20 km SW Manaus, Brazil, Asklepia demiti Erwin & Zamorano, sp. n., circa Rio Demiti, Brazil, Asklepia duofos Zamorano & Erwin, sp. n., 20 km SW Manaus, Brazil, Asklepia hilaris (Bates, 1871), comb. n, São Paulo de Olivença, Brazil, Asklepia grammechrysea Zamorano & Erwin, sp. n., circa Pithecia, Cocha Shinguito, Perú, Asklepia lebioides (Bates, 1871), comb. n, Santarém, Rio Tapajós, Brazil, Asklepia laetitia Zamorano & Erwin, sp. n., Leticia, Colombia, Asklepia matomena Zamorano & Erwin, sp.n., 20 km SW Manaus, Brazil; (pulchripennis species group) Asklepia adisi Erwin & Zamorano, sp. n., Ilha de Marchantaria, Lago Camaleão, Brazil, Asklepia asuncionensis Erwin & Zamorano, sp. n., Asunción, Río Paraguay, Paraguay, Asklepia biolat Erwin & Zamorano, sp. n., BIOLAT Biological Station, Pakitza, Perú, Asklepia bracheia Zamorano & Erwin, sp. n., circa Explornapo Camp, Río Napo, Cocha Shimagai, Perú, Asklepia cuiabaensis Erwin & Zamorano, sp. n., Cuiabá, Brazil, Asklepia ecuadoriana Erwin & Zamorano, sp. n., Limoncocha, Ecuador, Asklepia kathleenae Erwin & Zamorano, sp. n., Belém, Brazil, Asklepia macrops Erwin & Zamorano, sp. n., Concordia, Río Uruguay, Argentina, Asklepia marchantaria Erwin & Zamorano, sp. n., Ilha de Marchantaria, Lago Camaleão, Brazil, Asklepia marituba Zamorano & Erwin, sp. n., Marituba, Ananindeua, Brazil, Asklepia paraguayensis Zamorano & Erwin, sp. n., San Lorenzo, Rio Paraguay, Paraguay, Asklepia pakitza Erwin & Zamorano, sp. n., BIOLAT Biological Station, Pakitza, Perú, Asklepia pulchripennis (Bates, 1871), comb. n, Santarém, Rio Tapajós, Brazil, Asklepia samiriaensis Zamorano & Erwin, sp. n., Boca del Río Samiria, Perú, Asklepia stalametlitos Zamorano & Erwin, sp. n., Guayamer, Río Mamoré, Bolivia, Asklepia strandi Liebke, 1938, Guyana, Asklepia surinamensis Zamorano & Erwin, sp. n., l’Hermitage, Surinam River, Surinam, Asklepia vigilante Erwin & Zamorano, sp. n., Boca del Río Samiria, Perú. Images of adults of all 18 genera are provided.     

Choice of precursors for the measurement of protein turnover by the double-isotope method. Application to the study of mitochondrial proteins.

1. The double-isotope concept [Arias, Doyle & Schimke (1969) J. Biol. Chem. 224, 3303--3315] for the measurement of protein turnover was used to estimate the turnover of proteins in subcellular and submitochondrial fractions prepared from rat liver. 2. Double-isotope experiments with [3H]leucine as first precursor and [14C]leucine as second precursor were used to measure the turnover rates of proteins in subcellular and submitochondrial fractions. Solvent extraction procedures designed to remove lipids and nucleic acids from trichloroacetic acid precipitates only changed the isotope ratio of the microsomal fraction. It was not possible to measure turnover of proteins in mitochondrial and submitochondrial fractions with these precursors. 3. Double-isotope experiments were designed to minimize first-precursor reutilization by employing NaH14CO3. [3H]Arginine was used as second precursor. The turnover rates of protein in subcellular and submitochondrial fractions was measured. Solvent extraction procedures designed to remove lipids and nucleic acids showed changes in the isotope ratio for all subcellular fractions, especially in microsomal and detergent-soluble mitochondrial fractions. Isotope ratios of precipitates after solvent extraction indicate that, whereas considerable heterogeneity exists for the average rates of protein turnover in subcellular fractions, little heterogeneity is observed in the average rates of protein turnover in submitochondrial fractions.     

The molecular structure of a rapidly formed oligomeric adenosine tetraphosphate derivative from rat heart.

The inability to account for large systematic variations with time in soluble adenine nucleotides in perfused rat hearts [Bates, Perrett & Mowbray (1978) Biochem. J. 176, 485-493; Mowbray, Bates & Perrett (1981) FEBS Lett. 131, 55-59; Mowbray, Perrett & Bates (1984) Int. J. Biochem. 16, 889-894] led us to show that the soluble nucleotides are in rapid equilibrium with some hitherto unrecognized trichloroacetic acid/methanol-precipitable highly phosphorylated heteropolymeric form [Mowbray, Hutchinson, Tibbs & Morris (1984) Biochem. J. 223, 627-632]. Selective digestion coupled to chromatographic analysis together with m.s. and 31P-n.m.r. spectrometry have now been used to show that the likely structure for a purified oligomer that is in specific-radioactivity equilibrium with tissue ATP is 3-phospho-[glyceroyl-gamma-triphosphoroyl-5'-adenosine-3'-3- phospho]4 glyceroyl-gamma-triphosphoroyl-5'-adenosine.     

Sex- and phenotype-dependent metabolism of N tau-methylhistidine by mice.

The metabolism of N tau-[Me-14C]methylhistidine by mice was influenced by both the sex and the phenotype of the animal. After subcutaneous injection of labelled N tau-methylhistidine, recoveries of radioactivity in excreta were incomplete, and the distributions of radioactivity showed that different mouse phenotypes degraded N tau-methylhistidine in vivo to different extents. Hence the excretion of N tau-methylhistidine by mice cannot be used as an index of protein breakdown in vivo.     

Ligand-binding propeties of the muscarinic acetylcholine receptor in mouse neuroblastoma cells.

1. Muscarinic acetylcholine receptors in a plasma-membrane fraction derived from mouse neuroblastoma clone NIE-115 bind [3-3H]quinuclidinyl benzilate according to the Law of Mass Action (Kdissociation 40 pM, h0.96). 2. Antagonist and agonist binding to the receptor was studied by displacement of [3-3H]quinuclidinyl benzilate with non-radioactive ligands. The data show good agreement with similar data obtained on rat brain and ideal smooth muscle [Birdsall & Hulme (1976) J. Neurochem. 27, 7-16] indicating that the receptor is very similar in these three tissues.     

Application of topological methods in enzyme kinetics.

A criticism [Cornish-Bowden (1976) Biochem. J. 159, 167] of an algebraic method for deriving steady-state rate equations [Indge & Childs (1976) Biochem. J. 155, 567-570] is theoretically founded.     

An improved procedure, involving mass spectrometry, for N-terminal amino acid sequence determination of proteins which are N alpha-blocked.

A modification to a previously described procedure [Gray & del Valle (1970) Biochemistry 9, 2134-2137; Rose, Simona & Offord (1983) Biochem. J. 215, 261-272] for mass-spectral identification of the N-terminal regions of proteins is shown to be useful in cases where the N-terminus is blocked. Three proteins were studied: vesicular-stomatitis-virus N protein, Sendai-virus NP protein, and a rabbit immunoglobulin lambda-light chain. These proteins, found to be blocked at the N-terminus with either the acetyl group or a pyroglutamic acid residue, had all failed to yield to attempted Edman degradation, in one case even after attempted enzymic removal of the pyroglutamic acid residue. The N-terminal regions of all three proteins were sequenced by using the new procedure.     

The source of oxygen in the reaction catalysed by collagen lysyl hydroxylase.

The synthetic peptides (Pro-Pro-Gly)5 and (Ile-Lys-Gly)5-Phe were hydroxylated with collagen prolyl hydroxylase and lysyl hydroxylase in an 18O2 atmosphere. The oxygen atoms in the hydroxy groups of hydroxyproline and hydroxylysine were 87% and 6.5% respectively derived from the atmospheric 18O2. The results are consistent with those reported previously for proline hydroxylation in vivo [Fujimoto & Tamiya (1962) Biochem. J. 84, 333-335; Prockop, Kaplan & Udenfriend (1962) Biochem. Biophys. Res. Commun. 9, 192-196; Fujimoto & Tamiya (1963) Biochem. Biophys. Res. Commun. 10, 498-501; Prockop, Kaplan & Udenfriend (1963) Arch. Biochem. Biophys. 101, 499-503] and in vitro [Cardinale, Rhoads & Udenfriend (1971) Biochem. Biophys. Res. Commun. 43, 537-543] and for lysine hydroxylation in vivo [Fujimoto & Tamiya (1963) Biochem. Biophys. Res. Commun. 10, 498-501]. In view of the similarities of these two oxygenase-type hydroxylation reactions the participation of intermediates is proposed, the oxygen atoms of which are exchangeable with those of water. The atmospheric oxygen atoms incorporated into the intermediate must be equilibrated with water oxygen atoms in the slower lysyl hydroxylase reaction.     

Myofibrillar protein turnover. Synthesis of protein-bound 3-methylhistidine, actin, myosin heavy chain and aldolase in rat skeletal muscle in the fed and starved states.

The turnover of 3-methylhistidine (N tau-methylhistidine) and in some cases actin, myosin heavy chain and aldolase in skeletal muscle was measured in a number of experiments in growing and adult rats in the fed and overnight-starved states. In growing fed rats in three separate experiments, measurements of the methylation rate of protein-bound 3-methylhistidine by either [14C]- or [3H]-methyl-labelled S-adenosylmethionine show that 3-methylhistidine synthesis is slower than the overall rate of protein synthesis indicated by [14C]tyrosine incorporation. Values ranged from 36 to 51%. However, in one experiment with rapidly growing young fed rats, acute measurements over 1 h showed that 3-methylhistidine synthesis could be increased to the same rate as the overall rate. After overnight starvation in these rats, the steady-state synthesis rate of 3-methylhistidine was 38.8% of the overall rate. This was a similar value to that in adult non-growing rats, in which measurements of the relative labelling of 3-methylhistidine and histidine after a single injection of [14C]histidine indicated that 3-methylhistidine synthesis was 37% of the overall rate in the fed or overnight-starved state. According to measurements of actin, myosin heavy-chain and aldolase synthesis in the over-night-starved state with young rats, with a variety of precursors, slow turnover of 3-methylhistidine results from the specific slow turnover of actin, since turnover rates of myosin heavy chain, mixed protein and aldolase were 2.5, 3 and 3.4 times faster respectively. However, in the fed state synthesis rates of actin were increased disproportionately to give similar rates for all proteins. These results show that (a) 3-methylhistidine turnover in muscle is less than half the overall rate in both young and adult rats, (b) slow 3-methylhistidine turnover reflects the specifically slow turnover of actin compared with myosin heavy chain and other muscle proteins, and (c) during growth the synthesis rate of actin is particularly sensitive to the nutritional state and can be increased to a similar rate to that of other proteins.     

N6-Trimethyl-lysine metabolism. 3-Hydroxy-N6-trimethyl-lysine and carnitine biosynthesis.

Rats injected with N6-[Me-3H]trimethyl-lysine excrete in the urine five radioactively labelled metabolites. Two of these identified metabolites are carnitine and 4-trimethylammoniobutyrate. A third metabolite, identified as 5-trimethylammoniopentanoate, is not an intermediate in the biosynthesis of carnitine; the fourth and major metabolite, N2-acetyl-N6-trimethyl-lysine, is not a precursor of carnitine. The remaining metabolite (3-hydroxy-N6-trimethyl-lysine) is converted into trimethylammoniobutyrate and carnitine by rat liver slices and into trimethylammoniobutyrate by rat kidney slices. In rat liver and kidney-slice experiments, radioactivity from DL-N6-trimethyl-[1-14C]lysine and DL-N6-trimethyl-[2-14C]lysine was incorporated into N2-acetyl-N6-trimethyl-lysine and 3-hydroxy-N6-trimethyl-lysine, but not into trimethylammoniobutyrate or carnitine. A procedure was devised to purify milligram quantities of 3-hydroxy-N6-trimethyl-lysine from the urine of rats injected chronically with N6-trimethyl-lysine (100 mg/kg body wt. per day). The structure of 3-hydroxy-N6-trimethyl-lysine was confirmed chemically and by nuclear-magnetic-resonance spectrometry [Novak, Swift & Hoppel (1980) Biochem. J. 188, 521--527]. The sequence for carnitine biosynthesis in liver is: N6-trimethyl-lysine leads to 3-hydryxy-N6-trimethyl-lysine leads to leads to 4-trimethylammoniobutyrate leads to carnitine.     

Isotopic turnover rate and fractionation in multiple tissues of red rock lobster (Jasus edwardsii) and blue cod (Parapercis colias): Consequences for ecological studies

This study experimentally determined the turnover rates of δ¹³C and δ¹⁵N as a function of growth and metabolism and isotopic fractionation for different tissues in captive populations of red rock lobster (Jasus edwardsii) and blue cod (Parapercis colias). Isotopic turnover was estimated using the model of Hesslein et al. [Hesslein, R., Hallard, K., Ramlal, P., 1993. Replacement of sulfur, carbon, and nitrogen in tissue of growing broad whitefish (Coregonus nasus) in response to a change in diet traced by δ³⁴S, δ¹³C, and δ¹⁵N. Can. J. Fish. Aquat. Sci. 50, 2071–2076.]. Isotopic fractionations relative to diet differed among tissues and isotopes. Lobster muscle was more enriched than hemolymph and blue cod fin tissue was more enriched than blood for δ¹³C and δ¹⁵N. The metabolic component of turnover accounted for > 90% of the total isotopic turnover in lobster tissues and 30%–60% in blue cod tissues. Lobster muscle (half-life 147 d) and hemolymph (half-life 117 d) turnover rates were not significantly different but were faster than turnover rates of blue cod tissues. Whole blood, blood plasma fraction, and the blood cellular fraction had similar turnover rates; the whole blood half-life was 240 d for blue cod. Measuring turnover in larger, slower growing animals allowed for a more precise estimate of the metabolic component of isotopic turnover than in fast growing animals in which change is predominantly the result of dilution through growth. The differences in fractionation values among tissues observed here demonstrate that using generic trophic fractionation values would introduce error into diet reconstruction or migration studies. We demonstrate that a modified version of Hesslein et al.'s [Hesslein, R., Hallard, K., Ramlal, P., 1993. Replacement of sulfur, carbon, and nitrogen in tissue of growing broad whitefish (Coregonus nasus) in response to a change in diet traced by δ³⁴S, δ¹³C, and δ¹⁵N. Can. J. Fish. Aquat. Sci. 50, 2071–2076.] turnover model could be used to estimate the temporal component of migration.