Biomedical subjects

C A Slaughter

Publications and source records attributed to C A Slaughter.

At least 73 records · Page 4Linked to original sources

PubMed1993

Primary structure and topological analysis of a skeletal muscle-specific junctional sarcoplasmic reticulum glycoprotein (triadin).

The primary amino acid sequence for a highly abundant junctional sarcoplasmic reticulum glycoprotein (triadin) has been deduced from the cDNA sequence. Based on both biochemical analysis and the predicted amino acid sequence we suggest that this protein is an intrinsic membrane glycoprotein containing a single transmembrane domain that separates the protein into cytoplasmic and luminal domains. The cytoplasmic domain is proposed to contain the amino-terminal 47 amino acids. The remainder of the protein including the carboxyl terminus is proposed to be found within the lumen of the sarcoplasmic reticulum and contains an extremely high concentration of basic residues. Protease analysis of intact triads was consistent with the topological predictions. Western and Northern blots suggest that the protein is specifically expressed in skeletal muscle and not cardiac muscle or brain. The abundance and localization of this protein suggest that it plays an important regulatory or structural role in excitation-contraction coupling in skeletal muscle.

Amino Acid Sequence↗

PubMed1993

Induction of murine hepatic glutathione S-transferase by dietary dehydroepiandrosterone.

The naturally occurring steroid dehydroepiandrosterone (DHEA), when administered as a supplement to the diet of mice and rats, produces alterations in the relative concentrations of specific liver proteins; among these, a protein of Mr approximately 28 K is markedly induced by DHEA action. In the present study we identified the murine hepatic approximately 28 kDa protein as glutathione S-transferase subtype GT-8.7. Glutathione S-transferases belong to a gene superfamily that encode closely related proteins which are induced in liver and other tissues by various chemicals, including carcinogens and chemoprotective agents such as dietary antioxidants. Based on the above finding, we evaluated glutathione S-transferase activity in cytosols and microsomes prepared from liver tissue of mice fed either a control diet or a DHEA-containing diet (0.45%, by weight). The specific activity of hepatic cytosolic glutathione S-transferase in mice treated with DHEA up to 7 days was either unchanged or slightly decreased when compared to controls; however, treatment for 14 days or longer resulted in significant increases in activity. The specific activity of microsomal glutathione S-transferase also was increased by long-term DHEA treatment; however, its activity was approximately one-tenth of that in corresponding cytosols.

Administration, Oral↗

PubMed1993

Regulatory proteins of the proteasome.

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.

Adenosine Triphosphate↗

PubMed1992

Purification of component A of Rab geranylgeranyl transferase: possible identity with the choroideremia gene product.

Rab geranylgeranyl transferase (GG transferase) from rat brain contains two components, A and B. Component B comprises polypeptides of 60 and 38 kd. Here we report the purification of component A, a single 95 kd polypeptide. The holoenzyme attaches 3H-geranylgeranyl to cysteines in two GTP-binding proteins, Rab3A and Rab1A. The reaction is abolished when both cysteines in the COOH-terminal CysCys sequence of Rab1A are mutated to serines. The mutant protein inhibits transfer of 3H-geranylgeranyl to wild-type Rab1A and Rab3A, suggesting that the enzyme recognizes conserved sequences distinct from the COOH-terminus. Six peptides from rat component A show striking similarity to the product of the defective gene in choroideremia, an X-linked retinal degeneration disease. The choroideremia protein resembles Rab3A GDI, which binds Rab3A. We hypothesize that component A binds conserved sequences in Rab and that component B transfers geranylgeranyl. A defect in this reaction may cause choroideremia.

Alkyl and Aryl Transferases↗

PubMed1992

Identification and localization of a cysteinyl residue critical for the trypsin-like catalytic activity of the proteasome.

Chemical modification of the proteasome with N-ethylmaleimide (NEM) was performed for the purpose of identifying amino acid residues that play a role in the enzyme's proteolytic function. Modification of the proteasome with NEM specifically and irreversibly suppressed one of the three peptidase activities of the enzyme, viz., the "trypsin-like" activity. Leupeptin, a reversible competitive inhibitor of this activity, protected the activity from NEM inactivation, suggesting that NEM modifies a residue in the leupeptin binding site. Comparisons of enzyme samples labeled with [14C]NEM either in the presence or in the absence of leupeptin allowed the identification of a proteasome subunit containing an NEM-modified, leupeptin-protected cysteinyl residue. The leupeptin protection experiments suggest that residues of this subunit contribute to the active site responsible for the proteasome's trypsin-like activity. This subunit was purified by reverse-phase high-performance liquid chromatography. Peptide mapping and N-terminal amino acid sequencing were employed to acquire information about the primary structure of the subunit, including the sequence surrounding the leupeptin-protected cysteinyl residue. The sequencing data suggest that this proteasome subunit is evolutionarily related to other proteasome subunits that have been sequenced, which show no homology to other known proteases. The assignment of a catalytic function to a member of the proteasome family supports the hypothesis that proteasome subunits represent a structurally and possibly mechanistically novel group of proteases.

Amino Acid Sequence↗

PubMed1992

Autophosphorylation of skeletal muscle myosin light chain kinase.

Ca2+/calmodulin-dependent myosin light chain kinase phosphorylates the regulatory light chain of myosin. Rabbit skeletal muscle myosin light chain kinase also catalyzes a Ca2+/calmodulin-dependent autophosphorylation with a rapid rate of incorporation of 1 mol of 32P/mol of kinase and a slower rate of incorporation up to 1.52 mol of 32P/mol. Autophosphorylation was inhibited by a peptide substrate that has a low Km value for myosin light chain kinase. Autophosphorylation at both rates was concentration-independent, indicating an intramolecular mechanism. There were no significant changes in catalytic properties toward light chain and MgATP substrates or in calmodulin activation properties upon autophosphorylation. After digestion with V8 protease, phosphopeptides were purified and sequenced. Two phosphorylation sites were identified, Ser 160 and Ser 234, with the former associated with the rapid rate of phosphorylation. Both sites are located amino terminal of the catalytic domain. These results indicate that the extended "tail" region of the enzyme can fold into the active site of the kinase.

Adenosine Triphosphate↗

PubMed1992

Identification, purification, and characterization of a protein activator (PA28) of the 20 S proteasome (macropain).

A protein that greatly stimulates the multiple peptidase activities of the 20 S proteasome (also known as macropain, the multicatalytic protease complex, and 20 S protease) has been purified from bovine red blood cells and from bovine heart. The activator protein was a single polypeptide with an apparent molecular weight of 28,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and had a native molecular weight of approximately 180,000. This protein, which we have termed PA28, regulated all three of the putatively distinct peptidase activities displayed by each of two functionally different forms of the proteasome. This regulation usually included both an increase in the maximal reaction velocity and a decrease in the concentration of substrate required for half-maximal velocity and indicated that PA28 acted as a positive allosteric effector of the proteasome. PA28 failed, however, to stimulate the hydrolysis of large protein substrates such as casein and lysozyme. These results suggested that the hydrolysis of protein substrates occurred at a site or sites distinct from those that hydrolyzed small peptides and that the regulation of the two processes could be uncoupled. Evidence for direct binding of PA28 to the proteasome was obtained by glycerol density gradient centrifugation. PA28 may play an important regulatory role in intracellular proteolytic pathways mediated by the proteasome.

Allosteric Regulation↗

PubMed1992

Polyclonal antisera specific for the proenzyme form of each calpain.

Each subunit of calpain (EC 3.4.22.17) is proteolytically modified when the enzymes are exposed to calcium. These cleavages appear to be important for regulating the proteolytic activity and calcium-sensitivity of the proteinases. We have synthesized peptides that correspond to the sites of autoproteolytic modification within the catalytic subunit of each calpain. Polyclonal antisera raised against these peptides are highly specific for the unmodified catalytic subunit of each calpain. The antiserum specific for the N-terminal epitope of milli-calpain was used to demonstrate an inverse relationship between the presence of this N-terminal peptide and casein hydrolysis. The antiserum specific for the N-terminal epitope of micro-calpain was used to demonstrate proteolytic modification of the catalytic subunit of mu-calpain in rat erythrocytes treated with ionomycin and calcium.

Amino Acid Sequence↗

PubMed1992

Purification and characterization of a protein inhibitor of the 20S proteasome (macropain).

An inhibitory protein for the 20S proteasome (also known as macropain, the multicatalytic proteinase complex and 20S proteinase) has been purified from bovine red blood cells. The inhibitor has an apparent molecular weight of 31,000 on SDS-PAGE and appears to form multimers under nondenaturing conditions. This protein inhibited all three of the putatively distinct catalytic activities of proteasome A (the active form of the proteinase) characterized by the hydrolysis of synthetic peptides such as Z-VLR-MNA, Z-GGL-AMC or Suc-LLVY-AMC and Z-LLE-beta NA. The inhibitor also prevented the hydrolysis of large protein substrates such as casein, lysozyme and bovine serum albumin. Proteasome L (the latent form of the proteinase) does not degrade these large protein substrates, but does hydrolyze the three synthetic peptides at rates similar to those by proteasome A. The inhibitor inhibited only two of these peptidase activities of proteasome L (hydrolysis of Z-GGL-AMC and of Z-LLE-beta NA or Suc-LLVY-AMC); it had no effect on the hydrolysis of Z-VLR-MNA. The inhibitor was specific for inhibition of the proteasome and had no effect on the activity of any other proteinase tested including trypsin, chymotrypsin, papain, subtilisin and both isoforms of calpain. Kinetic analysis indicates that the inhibitor interacted with the proteasome by a mechanism involving tight-binding. Because the proteasome appears to be a key component of the ATP/ubiquitin-dependent pathway of intracellular protein degradation, the inhibitor may represent an important regulatory protein of this pathway.

Animals↗

PubMed1992

Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix.

The primary sequence of two components of the dystrophin-glycoprotein complex has been established by complementary, DNA cloning. The transmembrane 43K and extracellular 156K dystrophin-associated glycoproteins (DAGs) are encoded by a single messenger RNA and the extracellular 156K DAG binds laminin. Thus, the 156K DAG is a new laminin-binding glycoprotein which may provide a linkage between the sarcolemma and extracellular matrix. These results support the hypothesis that the dramatic reduction in the 156K DAG in Duchenne muscular dystrophy leads to a loss of a linkage between the sarcolemma and extracellular matrix and that this may render muscle fibres more susceptible to necrosis.

Amino Acid Sequence↗

PubMed1992

Fatty acid monooxygenation by P450BM-3: product identification and proposed mechanisms for the sequential hydroxylation reactions.

The soluble P450 isolated from Bacillus megaterium (the product of the CYP 102 gene) (P450BM-3) is a catalytically self-sufficient fatty acid hydroxylase which converts lauric, myristic, and palmitic acids to omega-1, omega-2, and omega-3 hydroxy analogs. The percentage distribution of the regioisomers depends on the substrate chain length. Lauric and myristic acids were preferentially metabolized to their omega-1 hydroxy counterparts while no hydroxylation occurred when capric acid was used as the substrate. Palmitic acid, when present at concentrations greater than the concentration of oxygen in the reaction medium (greater than 250 microM), was hydroxylated to its omega-1, omega-2, and omega-3 hydroxy analogs, with the percentage distribution of the regioisomers being 21:44:35, respectively. No omega hydroxylation of any of the fatty acids was detected. When the concentration of palmitic acid was less than the concentration of oxygen in the reaction mixture, it was noted that a number of additional products were formed. Under these conditions, unlike lauric and myristic acids, it was observed that palmitic acid was first converted to its monohydroxy isomers which were subsequently metabolized to a mixture of 14-ketohexadecanoic, 15-ketohexadecanoic, 13-hydroxy-14-ketohexadecanoic, 14-hydroxy-15-ketohexadecanoic, and 13,14-dihydroxyhexadecanoic acids with a relative distribution of 8:2:40:30:20, respectively. Thus, P450BM-3 is able not only to monohydroxylate a variety of fatty acids but also to further metabolize some of these primary metabolites to secondary and tertiary products. The present paper characterizes the products formed during the sequential hydroxylation of palmitic acid and proposes reaction pathways to explain these results.

Bacillus megaterium↗

PubMed1991

Molecular genetics of kinesin light chains: generation of isoforms by alternative splicing.

Movement of membrane-bounded organelles to intracellular destinations requires properly oriented microtubules and force-generating enzymes, such as the microtubule-stimulated ATPase kinesin. Kinesin is a heterotetramer with two heavy chain (approximately 124-kDa) and two light chain (approximately 64-kDa) subunits. Kinesin heavy chains contain both ATP- and microtubule-binding domains and are capable of force generation in vitro. Functions of the light chains are undetermined, although evidence suggests they interact with membrane surfaces. We have used molecular genetic approaches to dissect the kinesin light chain structure. Three distinct kinesin light chain cDNAs were cloned and sequenced from rat brain, and they were found to result from alternative splicing of a single gene. Polypeptides encoded by these cDNAs are identical except for their carboxyl ends. Synthesis of multiple light chains, differing from one another in primary structure, could provide a means of generating multiple, functionally specialized forms of the kinesin holoenzyme.

Amino Acid Sequence↗

PubMed1991

Expression and characterization of recombinant human acyloxyacyl hydrolase, a leukocyte enzyme that deacylates bacterial lipopolysaccharides.

The molecular cloning and eukaryotic cell expression of the complementary DNA for human neutrophil acyloxyacyl hydrolase (AOAH) are described. AOAH is a leukocyte enzyme that selectively removes the secondary (acyloxyacyl-linked) fatty acyl chains from the lipid A region of bacterial lipopolysaccharides (endotoxins), thereby detoxifying the molecules. The two disulfide-linked subunits of the enzyme are encoded by a single mRNA. The amino acid sequence of the protein contains a lipase consensus sequence in the large subunit and a region in the small subunit that is similar to the saposins, cofactors for sphingolipid hydrolases. The recombinant enzyme, like native AOAH, hydrolyzes secondary acyl chains from more than one position on the lipopolysaccharide backbone. Acyloxyacyl hydrolase is a novel two-component lipase that, by deacylating lipopolysaccharides, may modulate host inflammatory responses to Gram-negative bacterial invasion.

Acylation↗

PubMed1991

Mitochondrial import and processing of rat liver carnitine palmitoyltransferase II defines the amino terminus of the mature protein. Possibility of differential modification of the rat and human isoforms.

[35S]Methionine-labeled porcine heart citrate synthase (used here as a positive control) and rat liver carnitine palmitoyltransferase II (CPT II) were generated by in vitro transcription and translation of their cDNA constructs in appropriate Bluescript plasmids. Each product was imported into rat liver mitochondria in an energy-dependent manner to yield an immunoprecipitable protein of smaller size that comigrated with the corresponding purified enzyme. The size shift occurring with citrate synthase was consistent with the removal of the postulated 27-amino acid leader peptide. To determine the amino terminus of mature CPT II, [35S]methionine- or [3H]leucine-labeled material (after import and processing) was subjected to Edman degradation, followed by counting of the radioactivity released on each cycle. The results established that the precursor targeting peptide was cleaved between leucine 25 and serine 26 in the previously deduced amino acid sequence. Taken in conjunction with the recent report of Finocchiaro et al. (Finocchiaro, G., Taroni, F., Rocchi, M., Martin, A. L., Colombo, I., Tarelli, G. T., and DiDonato, S. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 661-665), the present results establish three key points concerning the rat and human forms of CPT II. First, it appears that in both species the initial translation product contains 658 amino acids and, upon mitochondrial import, is reduced in length by 25 residues through cleavage at an identical site. Second, the difference in electrophoretic mobility between the two mature proteins (documented earlier) presumably reflects either anomalous behavior of one of them on polyacrylamide gels or differential covalent modification. Finally, the recent suggestion by Brady et al. (Brady, P. S., Liu, J. S., Park, E. A., Hanson, R. W., and Brady, L. J. (1991) FASEB J. 5, A817) that our CPT II cDNA construct is incomplete in the 5'-coding region is refuted.

Amino Acid Sequence↗

PubMed1991

The primary structures of four subunits of the human, high-molecular-weight proteinase, macropain (proteasome), are distinct but homologous.

Macropain (proteasome) is a high-molecular-weight proteinase complex composed of at least 13 electrophoretically distinct subunits. Previous work, including peptide mapping and limited amino acid sequencing, suggested that most of the subunits belong to an evolutionarily related group of different gene products (Lee et al. (1990) Biochim. Biophys. Acta. 1037, 178-185). In order to define the extent and pattern of subunit relatedness, and to determine the structural basis for possible similarities and differences in subunit functions, we are deducing the primary structures of macropain subunits by cDNA cloning and DNA sequence analysis. We report here the primary structures of four subunits. The data clearly demonstrate that the proteins represent different, but homologous gene products. Surprisingly, no evidence for homology with any other protein, including proteinases, was obtained. These results suggest that macropain is comprised of a previously unidentified family of evolutionarily related polypeptides. Because biochemical data indicate that macropain contains several different proteinase activities, the current results raise the possibility that the macropain complex is composed of a group of novel proteinases, distinct from those of other structurally identifiable proteinase families.

Amino Acid Sequence↗

PubMed1991

Nonidentical subunits of p21H-ras farnesyltransferase. Peptide binding and farnesyl pyrophosphate carrier functions.

The protein farnesyltransferase purified from rat brain contains two nonidentical subunits, alpha and beta. The holoenzyme forms a stable complex with [3H]farnesyl pyrophosphate (FPP) that can be isolated by gel filtration. The [3H]FPP is not covalently bound to the enzyme; it is released unaltered when the enzyme is denatured. When incubated with an acceptor such as p21H-ras, the complex transfers [3H]farnesyl from the bound [3H]FPP to the ras protein. This transfer is not sensitive to dilution by unbound FPP, suggesting that the [3H]FPP is bound at a site that leads to direct transfer to the p21H-ras acceptor. Cross-linking studies show that the p21H-ras binds to the lower molecular weight subunit (beta-subunit), raising the possibility that the [3H]FPP binds to the alpha-subunit. If this suggestion can be confirmed, it would invoke a reaction mechanism in which the alpha-subunit acts as a prenyl pyrophosphate carrier that delivers FPP to p21H-ras which is bound to the beta-subunit.

Alkyl and Aryl Transferases↗

PubMed1991

Degradation of oxidized insulin B chain by the multiproteinase complex macropain (proteasome).

The peptides generated from the degradation of the oxidized B chain of bovine insulin by the multiproteinase complex macropain (proteasome) have been analyzed by reverse-phase peptide mapping and identified by N-terminal amino acid sequencing and composition analysis. Six of the 29 peptide bonds in the insulin B chain were found to be rapidly cleaved by macropain. The catalytic center that cleaves the Gln4-His5 bond could be distinguished from the center or centers that cleave the other preferred bonds by its specific susceptibility to inhibition by leupeptin, antipain, chymostatin, and pentamidine, suggesting that macropain utilizes at least two distinct catalytic centers for the degradation of this model polypeptide. The same effectors simultaneously enhance the rate of cleavage at the other susceptible sites in insulin B. The quantitative characteristics of this effect indicate that different catalytic centers of the complex may be functionally coupled, possibly by an allosteric mechanism or possibly by a mechanism in which binding to the catalytic centers is preceded by a rate-limiting binding of the substrate to a site or sites on the enzyme distinct from the catalytic centers. The kinetics of insulin B chain degradation indicate that macropain can catalyze sequential hydrolysis of peptide bonds in a single substrate molecule via a reaction pathway that involves channeling of peptide intermediates between different catalytic centers within the multienzyme complex. This capacity for channeling may confer potential physiological advantages of increasing the efficiency of amino acid recycling and reducing the pool sizes of peptide intermediates that are generated during the degradation of polypeptides in the intracellular milieu.

Amino Acid Sequence↗

PubMed1990

Sorting of peroxisomal membrane protein PMP47 from Candida boidinii into peroxisomal membranes of Saccharomyces cerevisiae.

A gene encoding PMP47, a peroxisomal integral membrane protein of the methylotrophic yeast Candida boidinii, was isolated from a genomic library. DNA sequencing of PMP47 revealed an open reading frame of 1269 base pairs capable of encoding a protein of 46,873 Da. At least two membrane-spanning regions in the protein are predicted from the sequence. Since the 3 amino acids at the carboxyl terminus are -AKE, PMP47 lacks a typical peroxisomal sorting signal. No significant similarities in primary structure between PMP47 and known proteins were observed, including PMP70, a rat peroxisomal membrane protein whose sequence has recently been reported (Kamijo, K., Taketani, S., Yokota, S., Osumi, T., and Hashimoto, T. (1990). J. Biol. Chem. 265, 4534-4540). In order to study the import and assembly of PMP47 into peroxisomes by genetic approaches, the gene was expressed in the yeast Saccharomyces cerevisiae. When PMP47 was expressed in cells grown on oleic acid to induce peroxisomes, the protein was observed exclusively in peroxisomes as determined by marker enzyme analysis of organelle fractions. Most of the PMP47 co-purified with the endogenous peroxisomal membrane proteins on isopycnic sucrose gradients. Either in the native host or when expressed in S. cerevisiae, PMP47 was not extractable from peroxisomal membranes by sodium carbonate at pH 11, indicating an integral membrane association. These results indicate that PMP47 is competent for sorting to and assembling into peroxisomal membranes in S. cerevisiae.

Amino Acid Sequence↗