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Biomedical subjects

P Christen

Publications and source records attributed to P Christen.

At least 145 records · Page 8Linked to original sources
PubMed1983

Crystalline aspartate aminotransferase: lattice-induced functional asymmetry of the two subunits.

The enzymic activity of crystalline mitochondrial aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) was determined in suspensions of noncrosslinked microcrystals in 30% (wt/vol) polyethylene glycol. The crystals (average dimensions, 22 x 5 x 0.8 micron) were small enough to preclude diffusional rate limitation. They had the same habit as the triclinic crystals used for the determination of the spatial structure of the enzyme by x-ray crystallographic analysis [Ford, G. C., Eichele, G., and Jansonius, J. N. (1980) Proc. Natl. Acad. Sci. USA 77, 2559-2563]. Determination of the Michaelis-Menten parameters showed that the packing of the enzyme dimer into the crystal lattice not only decreases its activity but also induces a functional nonequivalence of the two subunits that behave identically in solution. The crystalline enzyme possesses a high-affinity subunit with Km values similar to those of the enzyme in solution (K'm = 0.5 mM for aspartate and 1.2 mM for 2-oxoglutarate) and a low-affinity subunit (K'm = 5.5 mM and 14.5 mM, respectively). The catalytic activity of the high-affinity subunit is 3% and that of the low-affinity subunit is 15% of the activity of the enzyme in solution. The functional asymmetry of the crystalline enzyme dimer could also be demonstrated by selective mechanism-based modification of either type of active sites. In view of the apparently identical conformation of the two subunits in the crystalline enzyme, its decreased catalytic efficiency and its functional asymmetry likely are due to constraints exerted by the crystal lattice on the conformational adaptability of the two subunits. In triclinic crystals the two subunits of the enzyme dimer have dissimilar lattice contacts.

Animals↗
PubMed1982

Biosynthesis and topogenesis of aspartate aminotransferase isoenzymes in chicken embryo fibroblasts. The precursor of the mitochondrial isoenzyme is either imported into mitochondria or degraded in the cytosol.

In chicken embryo fibroblasts pulsed wih [35S]methionine, a precursor of mitochondrial aspartate aminotransferase with higher molecular weight (delta Mr approximately 3000) was detected by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide mapping of the precursor and the mature enzyme confirmed their precursor-product relationship. No precursor of the homologous cytosolic isoenzyme was found. The precursor of the mitochondrial isoenzyme is synthesized on membrane-free polysomes in the cytosol (Sonderegger, P., Jaussi, R., Christen, P., and Gehring, H. (1982) J. Biol. Chem. 257, 3339-3345); its half-life is 30 to 60 s. The pronounced susceptibility of the precursor toward exogenous proteases contrasts the stability of the mature enzyme and thus indicates that the conformation or the quarternary structure of the protein must change concomitantly with its import into mitochondria. Administration of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) to the cell cultures blocks the import of many matrix and inner membrane proteins into mitochondria. The precursor of mitochondrial aspartate aminotransferase is found to be accumulated in the cytosol. However, its steady state concentration in CCCP-treated cells exceeds the concentration in untreated cells by not more than 1 order of magnitude. During a chase, the radioactive precursor disappears with a half-life of approximately 5 with min without formation of mature enzyme. Thus, in CCCP-treated cells, a degradative process is limiting the accumulation of the precursor in the cytosol. When the chase is performed in the presence of cysteamine, an antagonist of CCCP, the precursor is processed to the mature enzyme. Newly synthesized cytosolic aspartate aminotransferase is not degraded.

Animals↗
PubMed1982

Chemical modification of a functional arginyl residue (Arg 292) of mitochondrial aspartate aminotransferase. Identification as the binding site for the distal carboxylate group of the substrate.

Mitochondrial aspartate aminotransferase is inactivated by dicarbonyl reagents selectively modifying arginyl residues. Treatment with phenylglyoxal inactivates the enzyme with concomitant modification of 2.7 mol of arginyl residues/mol of subunit. If the reaction is performed in the presence of the transaminating substrate pair aspartate/oxalacetate, only 1.3 mol of arginyl residues/mol of subunit are labeled and the enzymic activity remains at 75% of the original value. One particular residue, identified by peptide analysis as Arg 292, is completely protected against modification in the presence of the substrate pair, indicating a role of its guanidinium group in substrate binding. On the basis of x-ray crystallographic studies of the complex of apoenzyme with N-(5'-phosphopyridoxyl)-aspartate (minus pyridoxal form of the enzyme), Arg 292 has been proposed as the binding site of the distal carboxylate group (Ford, G. C., Eichele, G., and Jansonius, J. N. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2559-2563). The enzyme with blocked Arg 292 is not completely inactive, and its molecular activity toward dicarboxylic substrates is of the same order of magnitude as that of the native enzyme toward alanine, which is 10(5) times lower than that toward dicarboxylic substrates. The activity toward alanine is unchanged but the rate-enhancing effect of formate on the transamination of alanine is impaired. Formate is assumed to occupy the binding site of the distal carboxylate group (Morino, Y., Osman, A. M., and Okamoto, M. (1974) J. Biol. Chem. 249, 6684-6692). Apparently, the interaction of the distal carboxylate group of the substrate with Arg 292 underlies not only the binding specificity but also the kinetic specificity of aspartate aminotransferase for dicarboxylic substrates.

Aldehydes↗
PubMed1982

Biosynthesis of aspartate aminotransferases. Both the higher molecular weight precursor of mitochondrial aspartate aminotransferase and the cytosolic isoenzyme are synthesized on free polysomes.

The site of synthesis of the higher molecular weight precursor of mitochondrial aspartate aminotransferase (Sonderegger, P., Jaussi, R., and Christen, P. (1980) Biochem. Biophys. Res. Commun. 94, 1256-1260) has been determined by separation of free and membrane-bound polysomes under ionic conditions imitating the intracellular milieu and in vitro read-out translation of the two polysome fractions in a rabbit reticulocyte lysate. The amounts of the precursor of mitochondrial aspartate aminotransferase synthesized by free and membrane-bound polysomes were compared with the relative extent of the synthesized of cytosolic aspartate aminotransferase in the same fractions. Only a small (less than 10% of total) and for both isoenzymes quantitatively equivalent fraction was found to be produced by the membrane-bound polysome fraction; very likely, it has to be attributed to contaminating free polysomes. Apparently, the import of mitochondrial aspartate aminotransferase into the mitochondria does not involve an association of polysomes with intracellular membranes.

Animals↗
PubMed1982

Subcellular distribution of aspartate aminotransferase isoenzymes in chicken heart: quantitative study.

1. The content of the two aspartate aminotransferase isoenzymes in isolated mitochondria and in the cytosolic fraction from chicken heart was determined by radioimmunoassays. 2. The cationic isoenzyme was found to be associated with the mitochondrial fraction; its content measured in the cytosolic fraction was within the range of that of contaminating mitochondrial marker enzymes. 3. The anionic isoenzyme was found exclusively in the cytosolic fraction, in mitochondria a content of less than 0.05% of the total was measured. 4. Thus, in birds, the anionic and the cationic isoenzyme of aspartate aminotransferase show the same strict intracellular heterotopism as found previously in mammals.

Animals↗
PubMed1982

Measurement of prostatic acid phosphatase in serum and bone marrow: radioimmunoassay and enzymic measurement compared.

We quantitated the concentrations of prostatic acid phosphatases (EC 3.1.3.2) in serum and bone-marrow aspirates with three commercial radioimmunoassay kits, and the catalytic activities with a thymolphthalein monophosphate-based enzyme test. The enzyme's immunological activity in serum was compared with its catalytic activity for its potential as a detector of early prostatic cancer and its performance as an early marker of metastatic activity in bone. Neither measurement is useful for detecting early stages of prostatic cancer. The spread of carcinoma to lymph nodes or to bone is detected with greater frequency by radioimmunoassay than by the enzymic test. Radioimmunoassay also detected metastasis to the bone more frequently than did physical methods. Analytical and clinical performance of the four methods is described.

Acid Phosphatase↗
PubMed1981

Formation of hydroxypyruvaldehyde phosphate in human erythrocytes.

Human erythrocytes when incubated with [U-14C]glucose formed radioactively labeled hydroxypyruvaldehyde-P (CHOCOCH2OPO3(2-)). Using a "diagonal" chromatographic procedure on an anion exchange column for isolation and quantitative detection, up to 0.3 mumol of hydroxypyruvaldehyde-P/liter of packed cells was found. If glycolysis was blocked by inhibition of glyceraldehyde-3-P dehydrogenase with N-ethylmaleimide and iodoacetate, 0.7 mumol of hydroxypyruvaldehyde-P/liter of packed erythrocytes was detected. Hydroxypyruvaldehyde-P, found for the first time in eukaryotic cells, might be one of the long sought natural substrates of the glyoxalases.

Aldehydes↗
PubMed1981

Independent quantitation of the mitochondrial and the cytosolic isoenzyme of aspartate aminotransferase in chicken tissues by radioimmunoassays.

Two specific and sensitive radioimmunoassays for the independent determination of the cytosolic and mitochondrial isoenzyme of aspartate aminotransferase from chicken are described. The assays use 125I-labeled aspartate aminotransferases as tracer antigens, rabbit antisera, and goat anti-rabbit IgG antisera for indirect immunoprecipitation. The detection limit for the cytosolic isoenzyme was 177 pg and for the mitochondrial isoenzyme 83 pg. The assays were used for measuring the concentrations of the aspartate aminotransferase isoenzymes in serum, cultured fibroblasts, and various tissues of chicken. Both isoenzymes in the different tissue extracts proved immunologically identical with the corresponding isoenzymes isolated from heart.

Animals↗
PubMed1981

[Current assessment of radioimmunological determination (RIA) of prostate acid phosphatase in the diagnosis of prostatic cancer].

Sera of patients without any tumors of the prostate and sera of patients with benign and malignant tumors of the prostate were tested with the enzyme assay using thymolphthalein monophosphate as substrate. The results were compared with the RIA of Clinical Assay (Travenol) and found to yield an approximately 50% better recognition of malignant growth. However, false-positive rates were rather high (approximately 20%), which lowers the overall success rate to approximately 30%.

Acid Phosphatase↗
PubMed1980

Mitochondrial aspartate aminotransferase 27/32-410. Partially active enzyme derivative produced by limited proteolytic cleavage of native enzyme.

Native mitochondrial aspartate aminotransferase (AATase) is cleaved selectively by trypsin at the peptide bonds after Arg 26 or after Lys 31 yielding two shortened enzyme derivatives, AATase 27-410, and AATase 32-410. Recent x-ray crystallographic determination of the spatial structure of AATase has shown that the NH2-terminal segments of the two polypeptide chains of this dimeric enzyme pass in front of the active site clefts and form two separate junctions with the neighboring subunit which are not contiguous with the main subunit interface (Eichele, G., Ford, G. C., Glor, M., Jansonius, J. N., Mavrides, C., and Christen, P. (1979) J. Mol. Biol. 133, 161-180). The peptide bonds cleaved by trypsin are situated in the following stretch of the polypeptide chain which runs in exposed position on the surface of the subunit. The split-off peptide is lost during gel filtration. The molecular activity of AATase 27/32-410 (a mixture of about equal amounts of the two not readily separable derivatives) is about 3% of that of the native enzyme. In contrast, the K'm values for aspartate and 2-oxoglutarate are unchanged, indicating an unaltered geometry of the substrate binding site. A substantially diminished syncatalytic response of the reactivity of Cys 166 toward 5,5'-dithiobis-(2-nitrobenzoate) suggests that the decrease in catalytic activity is due to an interference with the syncatalytic conformational dynamics observed previously in AATase (Gehring, H., and Christen, P. (1978) J. Biol. Chem. 253, 3158-3163). Consonant with a role of the NH2-terminal segment in propagating the syncatalytic conformational rearrangements the rate of the tryptic cleavage is retarded 4-fold in the presence of the transaminating substrate pair aspartate and oxalacetate.

Amino Acids↗
PubMed1980

Production of glycolate by oxidation of the 1,2-dihydroxyethyl-thamin-diphosphate intermediate of transketolase with hexacyanoferrate(III) or H2O2.

In the presence of hexacyanoferrate(III), or other suitable oxidants, transketolase catalyzes the oxidative cleavage of its donor substrates xylulose 5-phosphate or fructose 6-phosphate into glycolate and glyceraldehyde 3-phosphate or erythrose 4-phosphate, respectively. Two moles of hexacyanoferrate(III) are reduced per mole of oxidatively cleaved donor substrate. In analogy to the oxidative trapping of carbanion intermediates of other enzymes [Healy, M. J. & Christen, P. (1973) Biochemistry, 12, 35-41], the kinetic features of the reaction indicate that the 1,2-dihydroxyethylthiamin diphosphate intermediate is the oxidation-susceptible species. The molecular activity for the oxidative cleavage of fructose 6-phosphate at a hexacyanoferrate(III) concentration of 0.5 mM is 0.2% of that for the normal transfer reaction with erythrose 4-phosphate as acceptor substrate. Glycolate is also produced with H2O2 as oxidant; however, the reaction is at least two orders of magnitude slower than with hexacyanoferrate(III).

Ferricyanides↗