A rapid assay for measuring the activity and the Mg2+ and Ca2+ requirements of phosphatidate phosphohydrolase in cytosolic and microsomal fractions of rat liver.

1. A rapid extraction and purification scheme was designed for the recovery of [3H]diacylglycerol formed during the assay of phosphatidate phosphohydrolase. 2. The importance of removing polyvalent cations, particularly Ca2+, from the phosphatidate and other reagents used in the assay of the phosphohydrolase activity was demonstrated. This was achieved mainly by treating the phosphatidate with a chelating resin and by adding 1 mM-EGTA and 1 mM-EDTA to the assays. 3. The activity of the phosphohydrolase in dialysed samples of the soluble and microsomal fractions of rat liver was very low. 4. Addition of optimum concentrations of MgCl2 resulted in a 110-167-fold stimulation in activity. 5. CaCl2 was also able to stimulate phosphohydrolase activity, but to a much smaller extent than MgCl2. 6. Chlorpromazine, an amphiphilic cation, inhibited the reaction when it was measured in these experiments by using a mixed emulsion of phosphatidylcholine and phosphatidate at pH 7.4. 7. Microsomal fractions that were preincubated with albumin contained very low activities of the Mg2+-dependent phosphohydrolase. When these were then incubated with the soluble fraction in the presence of oleate, the soluble phosphohydrolase attached to the microsomal membranes, and it retained its high dependency on Mg2+.     

Participation of the lone tryptophan residue of rat alpha-foetoprotein in its drug-binding sites. Comparison with rat serum albumin.

The participation in drug binding of the lone tryptophan residue of rat alpha-foetoprotein (alpha-FP) and serum albumin, the two main transport proteins of foetal serum, has been studied by two different techniques. Firstly, the effect on phenylbutazone and warfarin binding of the chemical derivatization of the lone tryptophan residue of both proteins by 2-nitrophenylsulphonyl chloride (NPS) was studied. Secondly, the effect of phenylbutazone binding on the intrinsic fluorescence of the tryptophan residue of rat alpha-FP and albumin was investigated. The specific modification of the proteins by NPS did not affect the binding of warfarin by rat alpha-FP and albumin, but greatly decreased the affinity of the high-affinity sites of rat alpha-FP for phenylbutazone, though the numbers of these sites were not significantly changed. However, for albumin a similar decrease in the affinity constant appeared to be due to the reaction conditions. The spectrofluorimetric studies showed that the lone tryptophan residue of alpha-FP and albumin was quenched by phenylbutazone binding, and the quenching paralleled the fractional saturation of the high-affinity site only in the case of albumin. The effect of phenylbutazone binding on the intrinsic fluorescence of rat alpha-FP indicated that the lone tryptophan residue of this foetal protein is not in the same molecular environment as that of albumin, not participating directly in the high-affinity site for phenylbutazone, and the effect may be via some induced conformational change in rat alpha-FP. These results also confirm our previous suggestion that the high-affinity sites for phenylbutazone and warfarin are different on the rat alpha-FP molecule. The results seem to indicate that this is also the case for albumin, but confirmation is necessary.     

Calcitonin receptors of human osteoclastoma

Osteoclast-rich cultures were prepared by disaggregation of osteoclastomas (giant cell tumour of bone) and settlement onto glass or plastic surfaces. Autoradiography using [125I]-salmon calcitonin ([125I]-sCT) revealed specific binding only to multinucleate giant cells (osteoclasts) and a minor population of mononuclear cells. [125I]-sCT competitive binding studies indicated a Kd of 5 x 10(-10) M and receptor number of approximately 1 million sites/osteoclast. sCT treatment resulted in a dose-dependent rise in cAMP (EC50 10(-10) M). Homogenates of an osteoclastoma also demonstrated specific binding of [125I]-sCT. Chemical cross-linking of a labelled synthetic sCT derivative. [125I]-[Arg11,18,Lys14]-sCT, using disuccinimidyl suberate, resulted in labelling of a receptor component of approximate Mr 85-90,000. The multinucleate giant cells (osteoclasts) of human osteoclastomas possess large number of CT receptors which exhibit the same binding kinetics and apparent Mr as those of other CT target cells.     

Determination of steady state and nonsteady-state glycerol kinetics in humans using deuterium-labeled tracer

Using deuterium-labeled glycerol as tracer and gas-liquid chromatography-mass spectrometry techniques for the determination of isotopic enrichment, we have developed a simple and ethically acceptable method of determining glycerol appearance rate in humans under steady-state and nonsteady-state conditions. In normal subjects, the appearance rate of glycerol in the post-absorptive state was 2.22 +/- 0.20 mumol X kg-1 X min-1, a value in agreement with those reported in studies with radioactively labeled tracers. The ratio nonesterified fatty acid (NEFA) appearance rate/glycerol appearance rate ranged from 1.95 to 3.40. In insulin-dependent diabetic patients with a mild degree of metabolic control, the appearance rate of glycerol was 2.48 +/- 0.29 mumol X kg-1 X min-1. The volume of distribution of glycerol, determined by the bolus injection technique, was (mean) 0.306 l X kg-1 in normal subjects and 0.308 l X kg-1 in insulin-independent diabetic patients. To evaluate the usefulness of the method for determination of glycerol kinetics in nonsteady-state conditions, we infused six normal subjects with natural glycerol and calculated the isotopically determined glycerol appearance rate using a single compartment model (volume of distribution 0.31 l X kg-1). During these tests, the expected glycerol appearance rates were successively 5.03 +/- 0.33, 7.48 +/- 0.39, 9.94 +/- 0.34, 7.48 +/- 0.39, and 5.03 +/- 0.33 mumol +/- kg-1 X min-1, whereas the corresponding isotopically determined appearance rates were 4.62 +/- 0.45, 6.95 +/- 0.56, 10.85 +/- 0.51, 7.35 +/- 0.34, and 5.28 +/- 0.12 mumol X kg-1 X min-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

A systematic study of the amino acid compositions and D/L ratios in fossil bones from two french neanderthalian sites

The reaction of racemization in which the L amino acids are reversibly converted into the corresponding D amino acids, proceeds in geological environment at such a slow rate that it may be used as a geochronometer. However, in fossils several parameters may affect the rate of racemization, i.e. moisture, surface, pH buffer and metal cations. This work consists of a systematic study of total amino acid content in fossil bones from two neanderthalian sites. The amino acid distributions of all specimens were determined and compared to that of fresh bone. The D/L amino acid were quantified and expressed in terms of age as a function of the temperature. The results led us to consider the «La Roquette» site older than «Les Canalettes» site.     

Time-resolved Raman spectroscopy with subpicosecond resolution: vibrational cooling and delocalization of strain energy in photodissociated (carbonmonoxy)hemoglobin

A Raman spectrometer that provides both subpicosecond resolution and independent, tunable pump and probe pulses is described. The spectrometer is employed to obtain time-resolved spectra of (carbonmonoxy)hemoglobin (HbCO) at times from 0.2 to 95 ps subsequent to ligand photodissociation. The spectra are interpreted in terms of a vibrationally hot heme that cools substantially in 10 ps. Concomitant with the proposed vibrational cooling is a slower relaxation, which we suggest results from a protein response to heme doming induced by ligand detachment. Results and interpretations are discussed in the context of current models of the heme photophysics and of hemoglobin reactivity.     

Carbon and nitrogen metabolism in ectomycorrhizal fungi and ectomycorrhizas

The literature concerning the metabolism of carbon and nitrogen compounds in ectomycorrhizal associations of trees is reviewed. The absorption and translocation of mineral ions by the mycelia require an energy source and a reductant which are both supplied by respiratory catabolism of carbohydrates produced by the host plant. Photosynthates are also required to generate the carbon skeletons for amino acid and carbohydrate syntheses during the growth of the mycelia. Competition for photosynthates occurs between the fungal cells and the various vegetative sinks in the host tree. The nature of carbon compounds involved in these processes, their routes of metabolism, the mechanisms of control and the partitioning of metabolites between the various sites of utilization are only poorly understood. Both ascomycetous and basidiomycetous ectomycorrhizal fungi synthesize and some, if not all, accumulate mannitol, trehalose and triglycerides. The fungal strains employ the Embden--Meyerhof pathway of glucose catabolism and the key enzymes of the pentose phosphate pathway (6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, transaldolase and transketolase). Anaplerotic CO2 fixation, via pyruvate carboxylase and/or phosphoenolpyruvate carboxykinase, provides high pools of amino acids. This process could be important in the recapture and assimilation of respired CO2 in the rhizosphere. The ectomycorrhizas are thought to contain the Embden--Meyerhof pathway, the pentose phosphate pathway and the tricarboxylic acid cycle, which provide the carbon skeletons for the assimilation of ammonia into amino acids. The main route of assimilation of ammonia appears to be through the glutamine synthetase-glutamate synthase cycle in the ectomycorrhizas. Glutamate dehydrogenase plays a minor role in this process. Glutamate dehydrogenase and glutamine synthetase are present in free-living ectomycorrhizal fungi and they participate in the assimilation of ammonia and the synthesis of amino acids through the glutamate dehydrogenase/glutamine synthetase sequence. In both in vitro cultures of fungi and ectomycorrhizas, the assimilated nitrogen accumulates in glutamine. Glutamine, but also ammonia, are thought to be exported from the fungal tissues to the host cells. Studies on the metabolism of ectomycorrhizas and ectomycorrhizal fungi have focused on the metabolic pathways and compounds which accumulate in the symbiotic tissues. Studies on regulation of the overall process, and the control of enzyme activity in particular, are still fragmentary.(ABSTRACT TRUNCATED AT 400 WORDS)     

Convergence to equilibrium in a genetic model with differential viability between the sexes

A single locus, diallelic selection model with female and male viability differences is studied. If the variables are ratios of allele frequencies in each sex, a 2-dimensional difference equation describes the model. Because of the strong monotonicity of the resulting map, every initial genotypic structure converges to an equilibrium structure assuming that no equilibrium has eigenvalues on the unit circle.Partially supported by funds provided by a Science and Education Grant to the USDA-Forest Service, Southeastern Forest Experiment Station, Population Genetics of Forest Trees Research Unit, Raleigh, USASupported by a grant from the Max Kade Foundation, New York, USA     

Purification and separation of two soluble fucosyltransferase activities of small intestinal mucosa

1. Rat small intestinal soluble fucosyltransferase is purified more than 2000-fold using chromatographic procedures with DEAE-cellulose, CM-cellulose, GDP-Sepharose and Concanavalin A-Sepharose. 2. Chromatography on Sephadex G15 of the final enzymatic fraction clearly separates two activities: a first peak incorporates fucose on asialoserotransferrin and a second peak on asialofetuin. 3. The use of small saccharidic acceptors (phenylgalactose, lactose, lacto-N-fucopentaose I) and the analysis of fucosylated asialoglycoproteins indicate that the first activity corresponds to an alpha-(3/4)-fucosyltransferase and the second one to an alpha-(1-2)-fucosyltransferase. 4. Protein analysis by polyacrylamide gel electrophoresis in the presence of SDS for each enzyme shows two bands corresponding to a mol. wt of about 65,000 and 70,000. The two enzymes have the same sensitivity to the action of N-ethylmaleimide.