Energy metabolism,body-temperature and breathing parameters in nontorpid blue-naped mousebirdsUrocolius macrourus

Summary Breathing frequencyF _r of resting blue-naped mousebirdsUrocolius macrourus lies between 50–70 per min and correlates directly with ambient temperatureT _a and energy metabolismM. The nocturnal mean energy intake per breath varies between 5.6–17.7 mJ/g. At highT _a the birds show gular fluttering with a relatively constantF _r of about 460 min^–1.M shows a constant absolute day-night difference of 25 J/g·h; the relative differences areT _a-dependent between 36–168% (lower values at lowerT _a). Thermal conductance is 2.10–2.15 J/g·h·°C (predicted 2.67), indicating a good insulation. Basal metabolic rate BMR is reduced by 63% compared to predicted values. At aT _a-range of +8–36 °C the birds are normothermic. Below this range nocturnalT _b andM decrease slightly with fallingT _a. The birds show partial heterothermia (shallow hypothermia). Clustering is an effective energy saving strategy which allows loweringM with keeping highT _b even at lowT _a.Oxygen-intake is controlled byF _r as well as by tidal volumeV t inT _a-dependent changing portions.V T can vary between 0.29–0.91 ml (mean value 49.7 ml).Abbreviations T _a ambient temperature - T _b body temperature - M energy metabolism - F _r breathing frequency - V T tidal volume - BMR basal metabolic rate - TNP thermoneutral point     

Abnormal expression ofα-L-fucosidase in lymphoid cell lines of fucosidosis patients

Fucosidosis is an autosomal recessive lysosomal storage disease due to a deficiency of-L-fucosidase activity in tissues and body fluids. Exponentially growing lymphoid cell cultures from four fucosidosis patients had 2.7-fold to 15.6-fold less extracellular-L-fucosidase protein and 28.8-fold to 144.0-fold less intracellular-L-fucosidase protein with negligible catalytic activity, compared to the mean of 19 control cultures. The percentage of total-L-fucosidase protein released extracellularly by cultures from the four patients was 64 to 85%, compared to 35±9% for control cultures. Intracellular and extracellular enzyme forms in fucosidosis and control cell lines were glycoproteins containing polypeptide chains ofM _r=52,000. During a 1.5-hr pulse-label with³⁵S-methionine,-L-fucosidase was synthesized by control cells and two fucosidosis cell lines as an intracellular form withM _r=58,000. During a subsequent 21-hr chase with unlabeled methionine, mutant enzyme was almost entirely processed to an extracellular form withM _r=62,000. In contrast, only 25–30% of control enzyme was processed to an extracellular form (M _r=62,000), with the remainder retained intracellularly (M _r=60,000). In the other two fucosidosis cell lines,-L-fucosidase was synthesized as an intracellular form withM _r=56,000 that was processed to an extracellular form withM _r=60,000. In summary, the fucosidosis mutation(s) affected the catalytic activity, quantity, and extracellular release of-L-fucosidase as expressed by lymphoid cells.This work was funded by NIH Grants DK 32161 to R. A. DiCioccio and GM 28428 to J. K. Darby.     

Effects of temperature and altitude on ventilation and gas exchange in chukars (Alectoris chukar)

Summary The effects of ambient temperature (T _a) on ventilation and gas exchange in chukar partridges (Alectoris chukar) were determined after acclimation to low and high altitute (LA and HA; 340 and 3,800 m, respectively).At both LA and HA, oxygen consumption ( ) increased with decreasingT _a atT _a from 20 to –20°C. AtT _a of 35 to 40°C, increased above thermoneutral values at HA but remained constant and minimal at LA. Water loss rates increased rapidly atT _a>30°C at both altitudes as birds began to pant. Ventilation rates (f) during panting were 5-to 23-fold greater than the minimalf at thermoneutralT _a.Increased atT _a below thermoneutrality was supported by increased minute volume (V i) at both altitudes. The change inV i was primarily a function of changing tidal volume (V t), althoughf increased slightly asT _a declined. Oxygen extraction ( ) remained fairly constant atT _a below 20°C at both altitudes. BothV t and were considerably lower when birds were panting than at lowerT _a.Chukars showed few obvious ventilatory adaptations to HA. The 35% change in between 340 and 3,800 m was accommodated by a corresponding change inV i (btps), most of which was accomplished by increasedf at HA, along with a slight increase in .Abbreviations and symbols HA high altitude - LA low altitude - rate of evaporative water loss - oxygen extraction efficiency - f respiratory frequency - V t tidal volume - V i minute volume - BMR basal metabolic rate - MHP metabolic heat production     

Solubilization and reconstitution of a vanadate-sensitive H+-ATPase from the plasma membrane ofBeta vulgaris

Summary A vanadate-sensitive H⁺-translocating ATPase isolated from red beet plasma membrane has been solubilized in active form and successfully reconstituted into artificial proteoliposomes. The H⁺-ATPase was solubilized in active form with deoxycholate, CHAPSO or octylglucoside in the presence of glycerol. Following detergent removal by gel filtration and reconstitution into proteoliposomes, ATP:Mg-dependent H⁺ transport could be measured as ionophore-reversible quenching of acridine orange fluorescence. Solubilization resulted in a three-to fourfold purification of the plasma membrane ATPase, with some additional enrichment of specific activity following reconstitution. H⁺ transport activity was inhibited half-maximally between 1 and 5 M vanadate (Na₃VO₄) and nearly abolished by 100 M vanadate. ATPase activity of native plasma membrane showed aK _i for vanadate inhibition of 9.5 M, and was inhibited up to 80% by 15 to 20 M vanadate (Na₃VO₄). ATPase activity of the reconstituted vesicles showed aK _i of 2.6 M for vanadate inhibition. The strong inhibition by low concentrations of vanadate indicates a plasma membrane rather than a mitochondrial or tonoplast origin for the reconstituted enzyme.     

Streptomycetes excreting dd-carboxypeptidases

Summary Excretion of exocellular dd-carboxypeptidases was tested using 128 strains of streptomycetes. Exocellular enzyme activity was shown in 13% of the trains investigated. Streptomyces strains showed low activity of excretion of dd-carboxypeptidases: 2.7–4.8 M of released C-terminal d-alanine (d-Ala) residue/1 culture supernatant per minute. Saccharopolyspora erythraea mutants produced considerably higher levels of exocellular enzymes, the dynamics of excretion depending upon the medium used. The highest activity of exocellular dd-carboxypeptidase production was 44 M d-Ala/1 culture supernatant per minute. The affinity of exocellular dd-carboxypeptidase of S. erythraea 64-575 for -lactam antibiotics was assessed by a statistical computer programme. The enzyme showed the lowest affinity for sodium cefotaxime, ID₅₀(M) = 7.5 × 10^–6, and the highest for potassium cephalosporin C, ID₅₀(M) = 5.0 × 10^–9, ID₅₀(M) representing the molar concentration of -lactan antibiotics which decreased by 50% the release of d-Ala. Offprint requests to: W. Kurzatkowski     

Characterization of a heat-stable NADP-dependent isocitrate dehydrogenase from the obligate thermophile Thermoleophilum minutum YS-4

Summary A thermostable NADP-dependent isocitrite dehydrogenase (IDH; EC. 1.1.1.42) was purified from the obligately thermophilic hydrocarbonoclastic bacterium Thermoleophilum minutum YS-4 (ATCC 35265). This was accomplished by affinity chromatography and electroelution from a nondenaturing polyacrylamide gel. The enzyme has an M _r of 60 000 and is composed of two identical subunits of M _r 30 500. The amino acid composition has an Arg/Lys ratio of 4:1 and very high levels of glycine. Under nondenaturing conditions, the enzyme has a distinct difference in electrophoretic mobility relative to IDHs obtained from other genera including the genus Thermus. The secondary strcuture consists of 16% -helix, 20% -sheet, 25% -turn and 37% random coil as determined by circular dichroism spectroscopy. The optimum pH and temperature for activity were 7.2 and 75° C respectively and the apparent K _mvalues for DL-isocitrate adn NADP⁺ were 33 M, and 48 M, respectively. The enzyme requires divalent cations, such as Mn²⁺ or Mg²⁺ for activity. NAD⁺ cannot substitute for NADP⁺. Oxaloacetate plus glyoxylate exert considerable inhibition on IDH activity while other glycolytic and tricarboxylic acid cycle intermediates have a lesser effect. p-Chloromercuribenzoic acid was inhibitory to the IDH although isocitrate and Mn²⁺ offered some protection from this inactivation. The enzyme is thermostable, retaining 84% and 57% of initial activity after incubation for 1 h at 60° and 70° C, respectively. Isocitrate provided protection from thermal inactivation allowing the IDH to maintain 21% activity after 1 h at 80° C. Offprint requests to: J. J. Perry     

Purification and properties ofl-serine dehydratase fromLactobacillus fermentum ATCC 14931

l-Serine dehydratase fromLactobacillus fermentum was purified 100-fold. It was stabilized by the presence of 1 mM l-cysteine in 50 mM phosphate buffer. M_r=150,000 was determined by gel filtration. The enzyme consists of four apparently identical subunits (M_r=40,000) that were observed after treatment with sodium dodecyl sulfate. The apparent K_m forl-serine was 65 mM. Fe⁺⁺ was required for the enzymatic activity, and the apparent K_m value for this reaction was 0.55 mM. Maximum enzymatic activity was observed at 45°C and pH 8.0 in 50 mM phosphate buffer. At pH values different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 11,400 and 22,800 cal × mol^–1 for temperature values more than and less than 35°C respectively. The purified enzyme showed a maximum absorption between 400 and 420 nm, indicating the presence of pyridoxal-5-phosphate (PLP) as a prosthetic group. The PLP concentration was 0.027 µmoles per milligram of protein. The data suggest that there is 1 mol of PLP for each protein subunit.     

Comparative Pharmacology of the Activity of Wild-type and G551D Mutated CFTR Chloride Channel: Effect of the Benzimidazolone Derivative NS004

The pharmacological activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel mutated at glycine 551 (G551D-CFTR) was studied in the presence of the benzimidazolone derivative NS004 and compared to that of wild-type (wt) CFTR. Using iodide (¹²⁵I) efflux and whole-cell patch-clamp techniques we found dose-dependent stimulation of phosphorylated wt-CFTR channels by NS004 with an EC ₅₀ 11 µM. With non-phosphorylated CFTR, the effect of NS004 was apparent only at concentration >100 µM. In G551D-CFTR-expressing CHO cells, neither forskolin (from 0.1 to 10 µM) nor NS004 (from 0.1 to 200 µM) added separately were able to stimulate channel activity. However, in the presence of 10 µM forskolin, NS004 stimulated G551D-CFTR activity in a dose-dependent manner with an EC ₅₀ 1.5 µM. We also determined the half-maximal effective concentration of forskolin (EC ₅₀ 3.2 µM) required to stimulate G551D channel activity in presence of 1.5 µM NS004. No inhibitory effect was observed at high concentration of NS004 with both wt- and G551D-CFTR. Whole-cell recordings of CFTR chloride currents from cells expressing wild-type or G551D-CFTR in the presence of NS004 were linear, time- and voltage-independent. The inhibitory profile of G551D-CFTR channel activity was similar to that of wild type, i.e., inhibition by glibenclamide (100 µM) and DPC (250 µM) but not by DIDS (200 µM) nor calixarene (100 nM). These results show that NS004 activates wt-CFTR channel and restores G551D-CFTR channel activity, the potency of which depends on both the concentration of NS004 and the phosphorylation status of CFTR.     

Ventilation and oxygen consumption in rosy finches and house finches at sea level and high altitude

Summary Rosy finches (Leucosticte arctoa) breed at altitudes above 3500 m in eastern California. House finches (Carpodacus mexicanus) belong to the same subfamily (Carduelinae), but breed at much lower elevations. Oxygen consumption ( ) and ventilatory parameters of these two species were measured over a wide range of ambient temperatures (T _a) at low altitude (LA; 150 m) and at high altitude (HA; 3800 m).Minimal nighttime 's of rosy finches and house finches at LA (T _a=30°C) were close to allometrically predicted values for passerine birds. At both altitudes, increased linearly with decreasingT _a betweenT _a=20 and –10°C. Resting 's were slightly higher at HA than at LA on average.In both species, minute volume ( ) was inversely related toT _a.T _a-correlated increases in resulted from significant increases in both ventilatory frequency (f) and tidal volume (V T) at both altitudes. Oxygen extraction efficiency ( ) was independent ofT _a in rosy finches at LA, but declined significantly with decreasingT _a in rosy finches at HA and in house finches at both altitudes.At a givenT _a, both species had significantly greater (BTPS) at HA than at LA. Altitude-correlated increases in resulted primarly from increases inf with little change inV T. was significantly greater at HA than at LA in both species.In spite of the difference in altitudinal distributions of rosy finches and house finches, there were few conspicuous interspecific differences in metabolic or ventilatory adaptation to altitude or lowT _a over the range of conditions examined.Symbols and abbreviations BMR basal metabolic rate - BTPS at body temperature and pressure, saturated - oxygen extraction efficiency - f ventilation frequency - h mean coefficient of heat transfer - HA high altitude - instantaneous oxygen consumption - LA low altitude - RH relative humidity - SMR standard metabolic rate - STPD standard temperature and pressure, dry - T temperature - a ambient - b body - lc lower critical of thermoneutral zone - minute volume - V T tidal volume     

Purification,characterization, and comparison of anα-glucosidase (endo-oligo-1, 4-glucosidase) from the mesophileBacillus subtilis and the obligate thermophileBacillus caldolyticus

Thep-nitrophenyl--d-maltoside hydrolyzing-glucosidase from the mesophileBacillus subtilis 25S and the obligate thermophileBacillus caldolyticus C2 was purified, characterized, and compared in order to determine the molecular mechanisms that may confer thermostability of starch-degrading enzymes. Both enzymes showed endo-oligo-1,4-glucosidase activity owing to their identical hydrolysis of linear malto-oligosaccharides to maltose and glucose as determined by thin-layer chromatography. Neither enzyme showed activity againstp-nitrophenyl--d-glucopyranoside, maltose, isomaltose, isomaltotriose, or panose. The enzymes may tentatively be classified as a panose-producing pullulanase owing to their hydrolysis of pullulan. The 25S and C2 enzymes were composed of two identical subunits of M_r 55,000 and 60,000 respectively. Both the 25S and C2 enzymes have a pI of 4.85, pH optimum of 7.5 and 7.0, and K_m values for the chromogenic substratep-nitrophenyl--d-maltoside of 2.96 mM and 1.31 mM respectively. The 25S enzyme exhibited optimal activity between 35 and 37°C, and complete inactivation after 10 min at 45°C, while the C2 enzyme showed optimal activity at 60°C and retained 100% of initial activity at 60°C for 2 h. The C2 enzyme required a minimum of 0.02% 2-mercaptoethanol or 0.01 mM EDTA for thermostability. A comparison of the amino acid compositions showed an increase in the number of proline, alanine, and leucine residues for the thermostable C2 enzyme. These alterations in hydrophobicity may influence enzyme thermostability; this may be a factor in the design of engineered proteins for industrial use.Florida Agricultural Experiment Station Journal Series No. 9985