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Bovine spleen Multicatalytic Proteinase Complex (Proteasome)
No full textAmino acid sequencing of subunits of the multicatalytic proteinase complex (MPC) isolated from bovine spleen showed an almost complete replacement of the X, Y, and Z subunits, constitutively expressed in most tissues, by the interferon-gamma-inducible LMP7, LMP2, and MECL1 subunits. A comparison with the pituitary MPC found a decreased chymotrypsin-like activity, a depressed peptidylglutamyl-peptide hydrolyzing activity, and a highly active component with properties similar to, but not identical with, that of the pituitary branched chain amino acid preferring (BrAAP) component. Unlike the pituitary BrAAP component, that of the spleen MPC exhibited a greatly decreased K-m, a highly increased catalytic efficiency (k(cat)), and a 80-180 times greater specificity constant (k(cat)/K-m) toward substrates with either branched chain or aromatic amino acid residues in the P-1 position. Also, unlike the pituitary BrAAP component, that of the spleen was sensitive to inactivation by 3,4-dichloroisocoumarin and sensitive to inhibition by peptidyl-aldehydes with either phenylalaninal or leucinal residues. Several phenylalaninal peptidyl-aldehydes were identified which selectively inhibited components of the spleen but not of the pituitary MPC. Two of the inhibitors are dipeptidyl-aldehydes, two others are tetrapeptidyl-aldehydes with a Pro residue in the P-3 position. The possibility is discussed that the properties and specificity of the spleen MPC are a consequence of the presence of the interferon-gamma-inducible subunits
Reactions of 14C-3,4-Dichloroisocoumarin with subunits of pituitary and spleen multicatalytic proteinase complexes (proteasomes)
No full textExposure to [C-14]-3,4-dichloroisocoumarin (DCI) of multicatalytic proteinase complexes (MPC) isolated from bovine pituitary and spleen leads to label incorporation into several beta-type subunits, to rapid inactivation of the chymotrypsin-like (ChT-L) activity, and to a slower inactivation of other activities of the MPC. The pituitary and spleen MPCs differ in that the first contains almost exclusively the X, Y, and Z subunits, whereas in the latter these subunits are largely replaced by LMP2, LMP7, and MECL1. Preincubation with two peptidyl aledhyde inhibitors of the ChT-L activity protected the X subunit in the pituitary MPC and unexpectedly the LMP2 subunit in the spleen MPC from label incorporation, despite the greater amino acid sequence homology of the LMP7 subunit to that of the X subunit, Losses in the yield of amino acids in both subunits, shown by amino acid sequencing, and lability of the DCI-protein bond indicated formation of an acyl derivative by reaction of DCI with the threonine OH group. Brief exposure to [C-14]-DCI led to preferential incorporation of label into the LMP2 and X subunits, consistent with the high inactivation rate constants of the ChT-L activity. Z-LLF-CHO, an inhibitor of ChT-L activity, but not Z-GPFL-CHO, an inhibitor of the branched chain amino acid preferring component, prevented incorporation of radioactivity into the X subunits, whereas both inhibitors prevented label incorporation into LMP2, indicating differences in susceptibility to inhibition between the two components. These and other data are consistent with involvement of the X and LMP2 subunits in expression of the ChT-L activity in the pituitary and spleen MPC, respectively, and suggest the catalytic functions of two other beta-subunits
Differences in Catalytic Activities and Subunit Pattern of Multicatalytic Proteinase Complexes (Proteasomes) Isolated from Bovine Pituitary, Lung and Liver
No full textPolyacrylamide gel electrophoresis and high performance liquid chromatography of multicatalytic proteinase complexes (MPC) isolated from bovine pituitary, lung and liver showed marked differences in the pattern of subunits, The concentrations of LMP7 in the lung and liver were 10 and 5 times, respectively, greater than those in the pituitary, whereas the chymotrypsin-like activity and the amount of a subunit (BO2), necessary for its expression, were markedly decreased in the lung and moderately decreased in the liver, Lower trypsinlike, small neutral amino acid preferring, and peptidyl-glutamyl-peptide hydrolyzing activities were also found in the lung and liver. The activity of the branched chain amino acid preferring component (BrAAP), predominantly latent in the pituitary, was highly activated in the lung and liver, as evidenced by a greatly decreased IT, and a 20-fold increase of the specificity constant V-max/K-m, indicating facilitated substrate access to its active site and increased affinity toward substrates with branched chain amino acids in the P-1 position, It is suggested that overexpression of LMP7 in the lung is related to increased exposure of the airways to foreign antigens, The possible association between amounts of LMP7 and the activation of the BrAAP component needs further examination
Isolation and N-Terminal sequence of multiple forms of granulins in human urine
No full textThree molecular forms of granulins (also known as epithelins) were isolated, for the first time, in human urine. Their N-terminal sequences, which have also been determined, are identical to those of granulins A and B, previously isolated from human leukocytes, and of granulin F, never isolated before but whose primary structure is known on the basis of the cDNA sequence. The urinary molecules, which show a molecular weight of about 6.5 kDa, are most likely produced by a posttranslational. proteolytic processing occurring at the level of the kidney, which appears to be the organ richest in granulin precursor mRNA. The molecular events underlying the precursor processing are unknown, even though the involvement of the protease kallikrein, an enzyme thought to be responsible for the processing of several polypeptidic growth factor precursors, could be hypothesized. Granulins, however, do not show antikallikrein activity. The presence in human urine of isoform F, previously not identified from other human sources, seems to support the hypothesis that mature forms of granulins are generated by an organ-specific precursor processing, on the basis of particular physiological requirements, and to suggest also that this isoform may play ''in vivo'' an important and specific role in the epithelial cells of the human kidney. (C) 1997 Academic Press
Proteinase inhibitors of the Kunitz family in fallow deer organs: a comparative study.
No full textProtein proteinase inhibitors belonging to the Kunitz family have been isolated and characterized in several fallow deer organs. In all the organs studied we found the basic pancreatic trypsin inhibitor (BPTI) while its isoforms, previously isolated and characterized in organs of other ruminant species (bovids and ovids), were absent. In the kidney, in addition to BPTI, active fragments of the inter-alpha-trypsin inhibitor were also isolated. The distribution of Kunitz-type inhibitors in different species of ruminants is compared and discussed on the basis of the expression of their encoding genes
The bovine lung 20S proteasome binding to reversible inhibitors: modulation by sodium ion
No full textThe effect of sodium ion on the inhibition exerted by Cbz-Leu-Leu-Leu-CHO on the chymotrypsin-like activity of the 20S proteasome isolated from bovine lung was investigated. The experimental data were analyzed using a standard linkage formalism. The calculated equilibrium affinity constants for the sodium ion binding to the free-enzyme and the inhibitor-bound enzyme are compatible to other well-characterized ion-involving heterotropic systems. The functional interdependence between the binding events played by the inhibitor and the sodium ion conforms to a heterotropic modulatory mechanism. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved
Rapid reverse phase-HPLC assay of HMG-CoA reductase activity.
Full text linkRadioisotope-based and mass spectrometry coupled to chromatographic techniques are the conventional methods for monitoring HMG-CoA reductase (HMGR) activity. Irrespective of offering adequate sensitivity, these methods are often cumbersome and time-consuming, requiring the handling of radiolabeled chemicals or elaborate ad-hoc derivatizing procedures. We propose a rapid and versatile reverse phase-HPLC method for assaying HMGR activity capable of monitoring the levels of both substrates (HMG-CoA and NADPH) and products (CoA, mevalonate, and NADP(+)) in a single 20 min run with no pretreatment required. The linear dynamic range was 10-26 pmol for HMG-CoA, 7-27 nmol for NADPH, 0.5-40 pmol for CoA and mevalonate, and 2-27 nmol for NADP(+), and limit of detection values were 2.67 pmol, 2.77 nmol, 0.27 pmol, and 1.3 nmol, respectively
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