Endocytosis of lactate dehydrogenase isoenzyme M4 in rats in vivo. Experiments with enzyme labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose.
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1. Pig lactate dehydrogenase isoenzyme M4 was labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose and injected intravenously into rats. Previous work has shown that this label does not influence the clearance of the enzyme (half-life about 26 min) and that it is retained within the lysosomes for several hours after endocytosis and breakdown of the protein [De Jong, Bouma & Gruber (1981) Biochem. J. 198, 45--51]. 2. The distribution of the radioactivity over a large number of tissues was determined 2 h after injection. A high percentage of the injected dose was found in liver (41%), spleen (10%) and bone including marrow (21%). 3. Autoradiography indicated uptake of the enzyme mainly by Kupffer cells of the liver, by spleen macrophages and by bone marrow macrophages. 4. Liver cells were isolated 1 h after injection of the enzyme. Kupffer cells, endothelial cells and parenchymal cells were found to endocytose the enzyme at rates corresponding to 4230, 35 and 25 ml of plasma/day per g of cell protein, respectively. 5. Previous injection of carbon particles greatly reduced the uptake of the enzyme by liver and spleen, but the uptake by bone marrow was not significantly changed. (+info)
Physicochemical properties and states of sulfhydryl groups of uricase from Candida utilis.
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Highly purified uricase [urate: oxygen oxidoreductase, EC 1.7.3.3] was obtained from Candida utilis by affinity chromatography with xanthine-agarose conjugate followed by chromatography with Sephadex G-200 in the presence of dithiothreitol. The uricase molecule had a molecular weight of 120,000 and was composed of four identical subunits with a molecular weight of 30,000. The amino acid composition was determined and the N-terminal amino acid was identified as methionine. Other physiochemical properties obtained were as follows; isoelectric point (5,6), alpha-helix content (12%; b0 = -76 +/- 15 degree), specific activity (25.8 units/mg protein) and Km value (1.0 x 10(-5) M) for uric acid at pH 8.5 and 25 degree C. The uricase preparation did not contain detectable amounts of the following metals; Fe, Cu, Mg, Mn, Co, Ni, Zn, and Cd. There were three cysteine residues in the subunit molecule and these were classified into two types by a chemical modification study; one is accessible to a reagent and the remaining two become accessible only after denaturation. The former may be located in the vicinity of the active center of the molecule, although it does not participate directly in the catalysis. (+info)
p-Cymene pathway in Pseudomonas putida: selective enrichment of defective mutants by using halogenated substrate analogs.
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Several classes of mutants of Pseudomonas putida (JT810) defective in the utilization of p-cymene as sole carbon source have been isolated. Selective enrichment of the mutants and for strains putatively cured of a degradative plasmid was achieved by incubation of cells in minimal growth media containing p-cymene (or p-cumate) and various halogenated analogs of the growth substrates or pathway intermediates. Analogs which led to successful enrichments included: p-chlorotoluene, p-bromotoluene, alpha-chloro-p-xylene, and p-iodobenzoate. A mutant strain, PpJT811, constitutive for the p-cymene pathway gave significantly greater enrichments of defective mutants than the wild-type parent PpJT810 after incubation with the halogenated analogs. It is suggested that the defective mutants are enriched because of the genetic alterations they possess, which confer immunity to a lethal synthesis performed by transformation of the analogs in clones possessing an intact p-cymene pathway. A nomenclature for the genetic organization of p-cymene pathway is described. (+info)
O-(4-Diazo-3,5-di[125I]iodobenzoyl)sucrose, a novel radioactive label for determining organ sites of catabolism of plasma proteins.
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A method is described for radiolabelling proteins with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose (DD125IBS). When proteins so labelled were degraded within lysosomes, the radioactive fragments were largely retained within the organelle. High specific radioactivities were obtained without changing the properties of the protein. The validity of the method was demonstrated in vivo in rats using the short-lived protein lactate dehydrogenase, isoenzyme M4, and the long-lived protein bovine serum albumin. Derivatization with DD125IBS did not alter the clearance of either protein. Uptake of DD125IBS-labelled lactate dehydrogenase, isoenzyme M4, by liver and spleen of rats was determined. Radioactivity in these tissues increased up to about 2 h after injection (at this time the protein has been almost completely cleared from the blood) and subsequently declined with a half-life of approx. 20 h. After differential fractionation of liver, radioactivity was largely found in the mitochondrial and lysosomal fraction. The results of these studies establish that DD125IBS covalently coupled to plasma proteins should be a useful radioactive tracer for identifying the tissue and cellular sites of catabolism of relatively long-lived circulating proteins. (+info)
Glyceraldehyde-3-phosphate dehydrogenase from human muscle: cleavage of tryptophanyl peptides with o-iodosobenzoic acid.
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The new procedure of protein fragmentation with o-iodosobenzoic acid (Mahoney & Hermodson, 1979; Biochemistry, 18, 3810-3814) was used to split three tryptophanyl peptide bonds in glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) from human skeletal muscle. The mixture obtained was separated into four homogeneous peptides by a one-step chromatographic procedure. High specificity of the cleavage reaction was proved by the amino acid analysis and determination of the N-terminal sequence of the peptide products. Both specificity ahd high yield obtained by this procedure confirmed its utility in the establishment of the protein primary structure. (+info)
Protein organization in Newcastle disease virus as revealed by perturbant treatment.
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Treatment of Newcastle disease virus with lithium diiodosalicylate differentially elutes the internally disposed proteins, M and NP, showing that these proteins are extrinsic, i.e., not associated with the lipid hydrophobic core. This selective elution requires disruption of the viral envelope, a process that is maximal at low temperature and influenced by the lipid composition of the virus envelope. (+info)
Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins.
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A rapid methof for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-idosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures. (+info)
Amino acid sequence of chymotrypsin inhibitor ECI from the seeds of Erythrina variegata (Linn.) var. Orientalis.
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The amino acids of the chymotrypsin inhibitor (ECI) from the Erythrina variegata seeds have been sequenced. The sequence was solved by analysis of peptides derived from the protein by enzymatic digestions with trypsin and Staphylococcus aureus V8 proteinase, as well as by chemical cleavage with o-iodosobenzoic acid. The ECI consists of 179 amino acid residues with a pyroglutamic acid as the N-terminal residue and has a calculated molecular weight of 19,791. Comparison of this sequence with the sequences of the two trypsin inhibitors, ETIa and ETIb, from the E. variegata seeds shows that about 60% of the residues of ECI are identical to those of ETIa and ETIb and that the reactive sites, Arg63, in ETIa and ETIb change to Leu64 in ECI. (+info)