Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Acetyl-CoA synthetase from bovine heart has been purified to homogeneity and been crystallized. The purification procedure involves ammonium sulfate precipitation and subsequent column chromatography on DEAE-Sepharose, Blue-Sepharose, CoA-Agarose and Superose 6. The purified enzyme has a specific activity of 45 units/mg protein, and its molecular weight estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis is approximately 72,000. The purified enzyme specifically utilizes acetate, ATP and CoA. Apparent Km values of the purified enzyme for acetate, CoA, and ATP were 0.16 mM, 0.14 mM and 0.25 mM, respectively. Limited digestion with trypsin, subtilisin BPN' and chymotrypsin revealed that the enzyme contains a 56 k segment resistant to these proteases. Secondary structure contents of the purified enzyme and the 56 k tryptic fragment were analyzed by circular dichroism measurement. The intact molecule contains 30% alpha-helix and 30% beta-structure, and trypsin digests alpha-helix rich regions more substantially. Western blot analysis of rat tissue homogenates by specific antibodies against the purified enzyme indicated that the 72 k enzyme is present in a wide variety of tissues and is most abundant in heart and kidney.
...
PMID:Kinetic properties and structural characterization of highly purified acetyl-CoA synthetase from bovine heart and tissue distribution of the enzyme in rat tissues. 761 Apr 60

It has recently been established that rat heart mitochondria contain two isoforms of carnitine palmitoyltransferase I (CPT I), the minor 88-kDa variant being identical to liver CPT I (L-CPT I) and the dominant 82-kDa form resembling the skeletal muscle enzyme (M-CPT I) (Weis, B. C., Esser, V., Foster, D. W., and McGarry, J. D. (1994) J. Biol. Chem. 269, 18712-18715). To quantify the functional contribution of L-CPT I to overall CPT I activity in heart mitochondria a selective inhibitor of the former was needed. The dinitrophenol analog of 2[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylic acid (etomoxir) (DNP-Et) was found to have this property. When liver and skeletal muscle mitochondria were exposed to DNP-Et in the presence of ATP and CoASH, the DNP-Et-CoA formed completely inhibited liver CPT I while leaving the muscle enzyme unaffected. Similar treatment of heart mitochondria blocked only the L-CPT I component. This had the effect of shifting the apparent Km for carnitine from approximately 200 to approximately 500 microM and the I50 value for malonyl-CoA (the concentration needed to suppress enzyme activity by 50%) from approximately 0.18 to approximately 0.06 microM, i.e. the heart system now behaved exactly the same as that from skeletal muscle. Taking the Km for carnitine of L-CPT I and M-CPT I to be 30 and 500 microM, respectively, it could be calculated that the former contributes approximately 2% to the total CPT I in heart. When the 82-kDa CPT I isoforms of heart and skeletal muscle were labeled with [3H]etomoxir and then exposed to trypsin, the fragmentation patterns obtained were identical and quite distinct from that given by CPT I from liver. We conclude that (i) DNP-Et, unlike other agents of the oxirane carboxylic acid class, has remarkable inhibitory selectivity for L-CPT I over M-CPT I; (ii) the previously puzzling observation that rat heart CPT I displays kinetic characteristics intermediate between those of the enzymes from liver and skeletal muscle is entirely accounted for by the low level expression of L-CPT I in the cardiac myocyte; and (iii) the dominant 82-kDa CPT I isoform in heart is identical to the muscle enzyme. The data reaffirm that, in contrast to CPT II, CPT I exists in at least two isoforms and that both are present in rat heart.
...
PMID:Use of a selective inhibitor of liver carnitine palmitoyltransferase I (CPT I) allows quantification of its contribution to total CPT I activity in rat heart. Evidence that the dominant cardiac CPT I isoform is identical to the skeletal muscle enzyme. 792 65

Transcarboxylase from Propionibacterium shermanii is a biotin-containing enzyme which catalyzes the reversible transfer of a carboxyl group from methylmalonyl-CoA to pyruvate. Transcarboxylase 26 S complexes consist of a central, hexameric 12 S subunit with 6 outer, 5 S subunits attached by 12 1.3 S biotinyl subunits. Each of the subunits has been cloned and expressed in Escherichia coli in active form. We have used the cloned genes in mutagenic studies of the structure-function interactions of these subunits. One particular target of our studies has been the evolutionarily conserved tetrapeptide Ala-Met-Bct-Met which surrounds the biotinyl lysine. We have investigated the properties of subunits containing leucine substitutions at each methionine (1.3 S M88L and 1.3 S M90L) by assaying their activity in the two partial reactions in which this subunit participates. Partial reaction assays demonstrate that leucine substitution at either position has a greater effect on the 12 S partial reaction than on the 5 S reaction and Met 88 is more significant catalytically than Met 90. To determine whether structural alterations in the 1.3 S mutants were responsible for the effects on activity, the conformations of these mutants were investigated. In vitro hydrolysis studies with trypsin and V8 protease demonstrated differences in the susceptibility of 1.3 S M88L relative to 1.3 S WT and 1.3 S M90L. Complexes of avidin with 1.3 S WT or mutant subunits, as monitored by fluorescence properties, indicated that the microenvironment of the biocytin of 1.3 S M88L was different from those of 1.3 S WT and 1.3 S M90L. By contrast, substrate binding (oxalacetate for 5 S and methylmalonyl-CoA for 12 S) was unaffected by any of the 1.3 S mutants. Taken together, these results indicate that the conserved tetrapeptide of the 1.3 S biotinyl subunit, particularly Met 88, is required to provide an essential conformation and proper binding properties for catalysis of the partial reactions and the overall reaction.
...
PMID:The conserved methionines of the 1.3 S biotinyl subunit of transcarboxylase: effect of mutations on conformation and activity. 834 13

Our objective was to isolate from rat liver mitochondria the malonyl-CoA-regulated and detergent-labile enzyme, carnitine palmitoyltransferase I (CPT I), whose properties and relationship to CPT II have been the subject of debate. After exposure of mitochondria to the dinitrophenol derivative of etomoxir-CoA (DNP-Et-CoA, a covalent inhibitor of CPT I), followed by detergent solubilization and blue Sepharose chromatography, the DNP-Et-labeled CPT I could be readily visualized on immunoblots using an anti-DNP monoclonal antibody. This material was used to raise a rabbit polyclonal antibody that recognized CPT I regardless of whether it was carrying a covalent ligand. Exposure of membranes from untreated mitochondria to a mixture of trypsin and chymotrypsin caused rapid loss of CPT I activity with a concomitant disappearance of immunodetectable protein. However, inclusion of malonyl-CoA in such incubations afforded major protection of CPT I activity. Under these conditions CPT I simply underwent truncation from approximately 90 to approximately 82 kDa. This was also true if CPT I had first been labeled with Et-CoA or DNP-Et-CoA prior to protease treatment. Thus, the presence of an inhibitor, whether reversible or irreversible, at the active site of CPT I limited the action of trypsin/chymotrypsin to removal of a small portion of the protein which was probably not necessary for catalytic function. These and other experiments with antibodies and proteases provided additional insight into the membrane topology of CPT I. They also strengthened our conviction that CPT I and CPT II are distinct proteins and that the former exists as tissue-specific isoforms. Finally, the 82-kDa truncated form of rat liver CPT I was isolated and subjected to partial amino acid analysis. Four unambiguous peptide sequences were obtained.
...
PMID:Inhibitors of mitochondrial carnitine palmitoyltransferase I limit the action of proteases on the enzyme. Isolation and partial amino acid analysis of a truncated form of the rat liver isozyme. 844 47

The enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT; EC 2.3.1.97) catalyses the transfer of myristic acid to the N-terminal glycine residue of cell and viral proteins. In this report the purification and partial sequencing of this enzyme from bovine brain is described. Using a combination of ammonium sulphate precipitation, chromatography on DEAE-Sepharose and affinity chromatography on CoA-agarose the enzyme was purified some 40-fold. Size-exclusion chromatography of this material in the presence of myristoyl-CoA yielded two peaks of enzyme activity with apparent molecular masses of 66 kDa and 43 kDa. Chromatography of the CoA-affinity-purified material on MONO-S followed by size-exclusion chromatography in the presence of myristoyl-CoA resulted in the isolation of the large form of the enzyme purified 3000-fold. Analysis by SDS/PAGE of this material showed a major 60 kDa silver-stained band. Similar analysis of the 43 kDa enzyme fraction from the same separation showed that this fraction contained several proteins including a major component with an apparent molecular mass of 49 kDa. Attempts at N-terminal sequencing of the 66 kDa form of the enzyme were unsuccessful and therefore this material was digested with trypsin and the resulting peptides separated by reverse-phase h.p.l.c. N-terminal protein sequencing of these peptides yielded sequences which show sequence similarity to those of yeast N-myristoyl-transferase.
...
PMID:Purification and partial sequencing of myristoyl-CoA:protein N-myristoyltransferase from bovine brain. 845 28

N-Myristoyl-CoA: protein N-myristoyltransferase (NMT) is the enzyme that catalyses the covalent transfer of myristic acid from myristoyl-CoA to the N-terminal glycine residue of a protein substrate. Subcellular fractionation of bovine brain indicates that NMT activity was located in both the cytosolic and the particulate fraction of the cell. Removal of the particulate fraction resulted in a 2-fold enhancement of NMT activity. Reconstitution of the particulate fraction and cytosolic fraction resulted in inhibition of the elevated cytosolic NMT activity. These results indicated the existence of putative inhibitor(s) activity of NMT located in the particulate fraction of bovine brain. The inhibitor was stable to heat and was identified as a protein, on the basis of its susceptibility to the proteases trypsin and chymotrypsin. Protease degradation first required the delipidation of the particulate fraction. The inhibitor was purified to near-homogeneity by heat treatment, solvent extraction and Sephacryl S-300 gelfiltration column chromatography. The inhibitor was purified 630-fold from the particulate fraction with a 20% yield. The protein inhibitor had an apparent molecular mass of 92 kDa by gel filtration and 71 kDa by SDS/PAGE, indicating the protein is monomeric. The inhibitor did not interact directly with myristoyl-CoA and possessed no protease, thioesterase or demyristoylase activity. Purified inhibitor protein inhibited the formation of 1167 pmol of myristoyl-peptide/min per mg of protein.
...
PMID:Identification, purification and characterization of a membrane-associated N-myristoyltransferase inhibitor protein from bovine brain. 848 42

Long-chain fatty acids and their acyl-CoA esters are potent inhibitors of nuclear thyroid hormone (T3) receptor in vitro. In the present study, we obtained evidence for acyl-CoA binding activity in the nuclear extract from rat liver. The activity sedimented at a position (3.5 S) identical with that of the T3 receptor, and the two activities sedimented together. Similarly, they coeluted on DEAE-Sephadex. After partial purification of the receptor, it was again inhibited strongly by acyl-CoAs. Heat stability and a partial trypsin digestion of the receptor both suggested that the action site of oleoyl-CoA overlapped the T3-binding domain of the receptor. In addition, thyroid hormone receptor beta 1, synthesized in vitro, bound oleoyl-CoA specifically and its T3-binding activity was inhibited. The dissociation constant for oleoyl-CoA binding to the partially purified receptor was 1.2 x 10(-7) M. This value as well as its molecular size distinguished the nuclear binding sites from the cytoplasmic fatty acid/acyl-CoA binding proteins. Oleoyl-CoA had no effect on the glucocorticoid receptor, another member of the nuclear hormone-receptor superfamily. From these results, we propose that thyroid hormone receptor is a specific acyl-CoA binding protein of the cell nucleus.
...
PMID:Fatty acyl-CoA binding activity of the nuclear thyroid hormone receptor. 849 47

The pyruvate dehydrogenase complex (PDC) has been purified to apparent homogeneity from the insect trypanosomatid, Crithidia fasciculata, a member of the most primitive eukaryotic group to contain mitochondria. Separation of the purified PDC by SDS-PAGE yielded five bands of 70 (p70), 60 (p60), 55, 46 and 36.5 kDa, which appeared to correspond to dihydrolipoyl dehydrogenase binding protein (E3BP), dihydrolipoyl transacetylase (E2), E3, E1 alpha and E1 beta, respectively. The purified complex did not exhibit endogenous PDHa kinase activity. p70 was much less abundant than p60. Polyclonal antisera raised against p70 did not cross-react with p60, and antisera raised against p60 did not cross-react with p70, suggesting that p60 did not arise from p70 by proteolysis. Both p70 and p60 contained similar amino terminal sequences. Both sequences contained the MPALSP motif similar to sequences present in both E3BP and E2 from other sources. Incubation of the purified PDC with [2-14C]pyruvate in the absence of CoA resulted in the acetylation of both p70 and p60, suggesting that both proteins contained lipoyl domains, but the specific incorporation of label into p70 was significantly greater than for p60. Limited proteolysis of the acetylated complex with trypsin yielded two major fragments derived from p60 of 35 and 30 kDa, corresponding to E2L and E2I, and one major acetylated fragment of 58 kDa derived from p70. Therefore, these results suggest that p70 is an E3BP and given its apparent M(r) and degree of acetylation, it contains multiple lipoyl domains.
...
PMID:Pyruvate dehydrogenase complex from the primitive insect trypanosomatid, Crithidia fasciculata: dihydrolipoyl dehydrogenase-binding protein has multiple lipoyl domains. 872 Jan 78

The mechanism by which long chain acyl-CoA (LC-CoA) esters affect the ATP-regulated potassium channel (KATP channel) was studied in inside-out patches isolated from mouse pancreatic beta cells. Addition of LC-CoA esters dramatically increased KATP channel activity. The stimulatory effect of the esters could be explained by the induction of a prolonged open state of the channel and did not involve alterations in single channel unitary conductance. Under control conditions, absence of adenine nucleotides, the distribution of KATP channel open time could be described by a single exponential, with a time constant of about 25 ms. Exposing the same patch to LC-CoA esters resulted in the appearance of an additional component with a time constant of >>150 ms, indicating a conformational change of the channel protein. LC-CoA esters were also able to potently activate channel activity at different ratios of ATP/ADP. Simultaneous additions of MgADP and LC-CoA esters resulted in a supra-additive effect on channel mean open time, characterized by openings of very long duration. Following modification of the KATP channel by a short exposure of the patch to the protease trypsin, the stimulatory effect of ADP on channel activity was lost while activation by LC-CoA esters still persisted. This indicates that LC-CoA esters and MgADP do not bind to the same site. We conclude that LC-CoA esters may play an important role in the physiological regulation of the KATP channel in the pancreatic beta cell by binding to a unique site and thereby inducing repolarization of the beta cell-membrane potential.
...
PMID:Evidence for a unique long chain acyl-CoA ester binding site on the ATP-regulated potassium channel in mouse pancreatic beta cells. 921 79

High-density lipoprotein (HDL) components remove cholesterol from cells by two independent mechanisms. Whereas HDL phospholipids pick up cholesterol that desorbs from the plasma membranes, HDL apolipoproteins appear to interact with cell-surface binding sites that target for removal pools of cellular cholesterol that feed into the cholesteryl ester cycle. Here we show that mild trypsin treatment of HDL almost completely abolishes this apolipoprotein-mediated cholesterol removal process. When HDL was treated with trypsin for various periods of time and then incubated with cholesterol-loaded fibroblasts, treatment for only 5 min reduced the ability of HDL to remove excess cholesterol from cellular pools that were accessible to esterification by the enzyme acyl CoA:cholesterol acyltransferase. This mild treatment digested less than 20% of HDL apolipoproteins and did not alter the lipid composition, size distribution, or electrophoretic mobility of the particles. Trypsin treatment of HDL for up to 1 h caused no further reduction in its ability to remove cellular cholesterol despite a greater than 2-fold increase in apolipoprotein digestion. Trypsin treatment of HDL also reduced its ability to deplete the cholesteryl ester content of sterol-laden macrophages. Promotion of cholesterol efflux from the plasma membrane by HDL phospholipids was unaffected by even extensive proteolysis. In parallel to the loss of cholesterol transport-stimulating activity, trypsin treatment of HDL for only 5 min nearly abolished its interaction with high-affinity binding sites on cholesterol-loaded fibroblasts. Reconstitution of trypsin-modified HDL with isolated apo A-I or apo A-II restored the cholesterol transport-stimulating activity of the particles. Thus a minor trypsin-labile fraction of HDL apolipoproteins is almost exclusively responsible for the apolipoprotein-dependent component of cholesterol efflux mediated by HDL particles.
...
PMID:Limited proteolysis of high density lipoprotein abolishes its interaction with cell-surface binding sites that promote cholesterol efflux. 921 13


<< Previous 1 2 3 4 5 6 7 8 9 Next >>

---