Enzyme & protein最新文献

This article reviews recent studies from our laboratory on protein regulators of the proteasome (multicatalytic proteasome complex) in red blood cells. A 240-kD inhibitory component (CF-2) exists in 26S proteasome complexes in a form which is conjugated to ubiquitin. Interestingly, this factor was shown to be identical to delta-aminolevulinic acid dehydratase (ALAD), involved in heme synthesis. A distinct 200-kD inhibitor of the proteasome is not present in the 26S complex. A 32-kD subunit of the 20S proteasome appears to be important for the latency of this core protease. Multiple isoelectric variants of the 32-kD subunit are consistent with phosphorylation. Another 20S proteasome subunit of 30 kD molecular weight is phosphorylated at specific serine residues by copurifying casein kinase II. It is suggested that ubiquitination and phosphorylation may account for at least part of the ATP dependency associated with the 26S proteasome complex. These modifications may play a role in the activity, assembly, translocation and/or turnover of this particle.

Recent studies have demonstrated that the proteasome, in addition to functioning in the complete degradation of cell proteins, is the source of most antigenic peptides presented to the immune system on major histocompatibility complex (MHC)-class I molecules. In this process, intracellular and viral proteins are degraded in the cytosol to 8- to 9-amino acid fragments, which are then transported into the endoplasmic reticulum, where they become associated with MHC-class I molecules and are thus delivered to the cell surface. A variety of evidence has shown that the proteasome and ATP-ubiquitin-dependent pathway are critical in this process: (1) In cells, selective inhibitors of proteasome function inhibit the bulk of protein degradation and thus prevent the generation of peptides necessary for class I presentation and the appearance of MHC on the cell surface. (2) Mutations that block ubiquitin conjugation prevent the generation of an antigenic peptide. (3) Modifications that lead to rapid degradation of a protein by the ubiquitin pathway enhance antigen presentation. (4) gamma-Interferon (gamma-IFN) induces new proteasome subunits, LMP2 and LMP7, encoded in the MHC region that are incorporated in place of constitutive proteasome subunits. Their incorporation does not affect rates of protein breakdown but causes changes in peptidase activities, i.e. they increase rates of cleavage after basic and hydrophobic residues and decrease cleavage after acidic residues. Transfections of cells with LMP2 or LMP7 cause similar changes in these peptidase activities as are caused by gamma-IFN. These modifications in peptidase activities should enhance the production of those types of peptides which are preferentially transported into endoplasmic reticulum and selectively bound to MHC-class I molecules.

Glucose-6-phosphate dehydrogenase Mediterranean (G6PD Med) is a common G6PD variant around the Mediterranean Sea characterized by severe enzyme deficiency and B-like electrophoretic mobility. The molecular basis of G6PD Med is a single C-->T transition at nucleotide (NT) 563. A polymorphic site exists in exon 11 of G6PD gene: in the wild-type NT 1311 is a C (1311C), but in some individuals from diverse populations a T (1311T) is present instead. Recent studies suggest that this C-->T transition is in linkage disequilibrium with G6PD Med genotype. In the normal population of Southern Italy (Sicily and Calabria) who is at risk for G6PD Med the NT 1311 allelic frequency is 16.7-20%; no data are available for the other regions. We screened the population of Marche region, Central Italy, in order to know the percentage of this polymorphism in an area not at risk for G6PD Med: we found that the 1311T frequency is about one half of that found in Southern Italy.

Short chain enoyl-CoA hydratase (SCEH) catalyzes the second step of the mitochondrial fatty acid beta-oxidation spiral. We isolated cDNA clones for human SCEH to facilitate investigation of the enzyme structure of the gene and to examine the genetic background of Reye's syndrome and sudden infant death. Oligo(dT)-primed and random primed human liver cDNA libraries in lambda gt11 were screened using the entire sequence of the rat SCEH cDNA as a probe. Three positive clones covered the full-length cDNA sequence with an open reading frame encoding a precursor polypeptide of 290 amino acid residues, and deduced relative molecular mass (31,280) with a putative N-terminal presequence of 29 residues, a 5'-untranslated sequence of 21 bp and a 3'-untranslated sequence of 391 bp. Comparison with the rat SCEH cDNA showed that the deduced amino acid sequence of the human SCEH precursor is 84% identical to that of the rat enzyme precursor. Northern blot analysis gave a single mRNA species of 1.6 kb in the human liver, fibroblast and muscle.

The modulation of the intracellular glucocorticoidal effect on surfactant synthesis of the fetal lung by the metabolic capacity of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) could be an important factor in lung maturation. The kinetic properties of microsomal 11 beta-HSD of the rat lung are characterized with respect to product inhibition, substrate specificity, effect of electrolytes or trace elements, and the dependence of the oxidase reductase (OR) ratio on incubation conditions. With NADP+ product inhibition of the reductase was demonstrated. The most common trace elements and electrolytes exhibited no effect on the activity of 11 beta-HSD. It is shown that the OR ratio was strongly dependent on assay conditions. With optimal assay conditions oxidase activity exceeds reductase activity in adult and fetal rat lung microsomes (OR ratio > 1). Thus, glucocorticoids are mainly metabolized to their inactive forms. The enzyme activity in the adult is about 10 times higher than in the fetal lung. The low enzyme activity in fetal lungs could be the reason why the glucocorticoidal effects on surfactant synthesis are not suppressed despite the predominance of oxidase activity.

A myeloma cell line (KHM-4) from a patient with multiple myeloma and idiopathic hyperammonemia, and another myeloma cell line (RPMI8226) were seen to have activity to form ammonia from arginine. High activity of ornithine transcarbamylase (OTC), a hepatic urea cycle enzyme, was detected in these cell lines. OTC of these cells was much more heat-stable than the liver enzyme, and did not cross-react with an antibody against the liver enzyme. As the OTC activity was also detected in the culture medium of the myeloma cells and because the activity was markedly decreased by the antimycoplasma drug MC-210, the OTC activity was assumed to be associated with mycoplasma infection. Polymerase chain reaction, using degenerate oligonucleotide mixtures corresponding to the two highly conserved sequences of OTC, amplified a DNA sequence that apparently encodes a portion (about 67% in length) of mycoplasma OTC. The predicted amino acid sequence of the mycoplasma enzyme was 33-47% identical with those of the enzymes of bacteria, yeast and mammals.

Eukaryotic proteasomes are unusually large protein complexes with characteristic sets of subunits and have been classified into two isoforms with apparent sedimentation coefficients of 20S and 26S, respectively. The 20S proteasome (previously named the multicatalytic proteinase complex) is a cylindrical particle with a molecular weight (MW) of approximately 750 kD. It is a dimeric assembly of two symmetrical discs, each consisting of 7 alpha-type subunits and 7 beta-type subunits, having the molecular organization alpha n[1-7)beta n[1-7)beta n[1-7)alpha n[1-7), where 'n' indicates the number of heterogeneous 7 subunits with MWs of 21-32 kD. The alpha-type and beta-type subunits constitute a unique multi-gene family encoding previously unidentified, but homologous, polypeptides that have been conserved during evolution. Interestingly, some beta-type subunits with catalytic functions appear to be replaced by very homologous, but distinct, gene products that might be generated by gene duplication in response to extracellular signals, such as gamma-interferon, suggesting that the 20S proteasome exists in cells as a heterogeneous population with functional diversity. The 26S proteasome is a eukaryotic ATP-dependent protease, selectively degrading various cellular proteins with specific degradation signals such as a multi-ubiquitin chain. It is a cylindrical caterpillar-shaped complex with a MW of about 2,000 kD. The 26S proteasome is a symmetrical assembly of a central 20S proteasome and a large terminal polypeptide complex with an apparent sedimentation coefficient of 22S. The terminal 22S subset consists of multiple components with MWs of 30-110 kD, which possibly have regulatory functions, and contains multiple ATPases, a de-ubiquitinating enzyme and the recognition molecule(s) for the target proteins. Thus the 26S proteasome is a multi-molecular assembly, consisting of the 20S proteasome and the 22S regulatory subunit complex.

The function of the proteasome is controlled by a variety of specific regulatory proteins including activators, inhibitors, and modulators. Two recently discovered activators, termed PA28 and PA700, bind to the terminal rings of the proteasome to form proteasome-regulatory complexes which display greatly increased proteolytic activity. PA28 is a high-affinity activator of the proteasome's multiple peptidase activities. The carboxyl terminus of PA28 is required for its binding to the proteasome. PA700 binds to the proteasome via an ATP-dependent mechanism. PA700 has ATPase activity, and at least four of PA700's 16 subunits are members of a protein family containing a concensus sequence for ATP binding. Proteasome-PA700 complexes are activated with respect to both the hydrolysis of peptide substrates and the hydrolysis of ubiquitinated proteins.

The multicatalytic proteinase complex (MPC), also called the proteasome, is a ubiquitous particle (19S) that is required for life. It is found in the cytoplasm and nucleus of all eukaryotic cells where it degrades selected cytosolic and nuclear proteins. It forms the proteolytic core of the 26S complex that represents the final step in the ubiquitin-dependent pathway of proteolysis. The MPC expresses at least five distinct proteolytic activities. Three activities preferring cleavages on the carboxyl side of neutral amino acids were described: an activity cleaving after branched chain residues, termed branched chain amino acid preferring, that is a major factor in the degradation of proteins, an activity preferring cleavages after bulky hydrophobic residues designated chymotrypsin-like, and an activity cleaving between small neutral amino acids. Activities cleaving after basic (trypsin-like) and acidic residues (peptidylglutamyl peptide-hydrolyzing) have also been described. The expression of multiple proteolytic activities with diverse specificities may provide a functional advantage that allows efficient hydrolysis of target proteins.

Two major 2-aminoadipate aminotransferase (AadAT) activities of human liver extract were separated by DEAE-Sepharose column chromatography. The faster eluting enzyme was designated AadAT-I and the other one AadAT-II. AadAT-I had a hgih Km value for aminoadipate, 20 mmol/l, and a low Km value for glutamate, 1.4 mmol/l. In contrast, AadAT-II had a low Km value for aminoadipate, 0.25 mmol/l, and a high Km value for glutamate, 12.5 mmol/l. AadAT-I and AadAT-II were mainly localized in the supernatant and mitochondrial fraction, respectively. AadAT-I demonstrated only glutamate-2-oxoadipate or 2-aminoadipate-2-oxoglutarate aminotransferase activities. AadAT-II further showed the activity of tryptophan and kynurenine. On the basis of Km values and subcellular localization of the isoenzymes, a plausible role was suggested for them involving the metabolism of lysine and tryptophan.