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

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

We have investigated the localization of palmitoyl-CoA (hexadecanoyl-CoA) synthetase (EC 6.2.1.3) and cerotoyl-CoA (hexacosanoyl-CoA) synthetase in peroxisomes isolated from rat liver. Palmitoyl-CoA and cerotoyl-CoA synthetases, like acyl-CoA: dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42), are present in the peroxisomal membrane. Trypsin treatment of intact peroxisomes led to the disappearance of both palmitoyl-CoA and cerotoyl-CoA synthetase activities but had little, if any, effect on L-alpha-hydroxy-acid oxidase (EC 1.1.3.15), D-amino acid oxidase (EC 1.4.3.3) or acyl-CoA:dihydroxyacetone phosphate acyltransferase. The latter three enzymes were inactivated if the trypsin treatment was preceeded by disruption of the peroxisomes by sonication. These results show that the active site, or at least domains essential for the activity of cerotoyl-CoA synthetase, like that of palmitoyl-CoA synthetase, is located on the cytosolic face of the peroxisomal membrane.
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PMID:Topography of very-long-chain-fatty-acid-activating activity in peroxisomes from rat liver. 182 48

Native rat liver methylmalonate semialdehyde dehydrogenase was proteolyzed by lysylendopeptidase C, chymotrypsin, and trypsin to generate different cleavage fragments of molecular masses: 50, 8, 55, 44, 39, 53, 45, and 40 kDa. A proteolytic cleavage map of MMSDH was constructed based on sequencing data and a comparison of appearance and degradation rates of the different protein fragments as shown by SDS-PAGE. NAD+ was highly effective as a protector against proteolysis in both the N-terminal and the C-terminal parts of the intact enzyme. NADH did not efficiently protect the intact enzyme; however, it stabilized proteolytic fragment L50 from further degradation. This suggests that the NAD(+)-binding domain is not destroyed by cleavage of the N-terminal part of MMSDH. CoA had no effect on the proteolytic cleavage patterns of MMSDH. However, CoA esters reduced the protective effect of NAD+ with an order of effectiveness of acetyl-CoA greater than propionyl-CoA greater than butyryl-CoA. p-Nitrophenyl acetate, substrate for esterase activity by the enzyme, partially prevented the protective effect of NAD+ against proteolysis. These results suggest that S-acylation of the enzyme prevents a stabilizing conformational change induced in MMSDH by NAD+ binding.
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PMID:The effect of ligand binding on the proteolytic pattern of methylmalonate semialdehyde dehydrogenase. 189 92

Pyruvate:NADP+ oxidoreductase from Euglena gracilis, a homodimeric protein with a molecular weight of 309 kDa, is an iron-sulfur flavoenzyme that contains thiamin pyrophosphate (TPP). The functional structure of the enzyme was studied by a limited proteolysis experiment using trypsin. The evidence obtained shows that the enzyme consists of two functional domains, one of which contains an iron-sulfur cluster, which can be isolated as a homodimeric fragment of approximately 220 kDa by proteolysis. The other domain that contains FAD is released as a monomeric fragment of approximately 55 kDa. The pyruvate dehydrogenase reaction is still catalyzed by the large fragment when NADP+ is substituted by methyl viologen, while the small fragment retains a diaphorase-like electron-transfer activity from NADPH to MV. It is thus shown that pyruvate is oxidized in a CoA-dependent reaction to form CO2 and acetyl-CoA in the iron-sulfur domain, and that the two electrons formed are transferred to the FAD domain in which NADP+ is reduced. TPP is considered to be associated in the iron-sulfur domain. The NH2-terminal sequences of the enzyme and its proteolytic fragments reveal that the iron-sulfur domain occurs in the NH2-terminal side of the enzyme. For elucidation of the O2 instability of the enzyme, limited proteolysis was attempted in air. The tryptic fragment derived from the iron-sulfur domain, similar to the native enzyme, appears to be inactivated by direct contact with O2. In contrast, the FAD domain, when separated from the other domain, is quite stable in air, although the diaphorase activity decays when the native enzyme is exposed to O2.
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PMID:Pyruvate:NADP+ oxidoreductase from Euglena gracilis: limited proteolysis of the enzyme with trypsin. 191 Feb 87

Upon differential centrifugation of guinea pig intestine mucosal cells homogenate, fatty acyl-CoA:NADPH oxidoreductase (long chain alcohol forming) was found to be enriched in the light mitochondrial (L) fraction (sedimenting between 66,000 x g min and 500,000 x g min) which contained mainly mitochondria, lysosomes, and peroxisomes. Peroxisomes (marker enzymes: catalase and dihydroxyacetone phosphate acyltransferase) present in the L fraction were separated from other organelles in a Nycodenz density gradient centrifugation employing a vertical rotor. By comparing the distribution of acyl-CoA reductase with different marker enzymes in the gradient, it was concluded that this reductase is primarily localized in the microperoxisomes (microbodies). The topography of the membrane-bound enzyme in the isolated organelles was studied by checking its lability toward trypsin in the absence and presence of the detergent Triton X-100. The results suggested that acyl-CoA reductase is localized on the outer surface (cytosolic side) of microperoxisomal membrane.
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PMID:Peroxisomal localization of acyl-coenzyme A reductase (long chain alcohol forming) in guinea pig intestine mucosal cells. 206 6

1. The enzymatic mechanism of the alpha-hydroxylation of lignoceroyl-CoA, an intermediate in the synthesis of hydroxyceramide, was studied. In the presence of NADPH, sphingosine and microsomes from 20-day-old rat brain, 14C from [1-14C]lignoceroyl-CoA was incorporated into hydroxyceramide. 2. The alpha-hydroxylation of lignoceroyl-CoA in rat brain microsomes was strongly inhibited by a rabbit anti-immunoglobulin G which was prepared against rat liver microsomal NADPH-cytochrome c reductase. However, anti-immunoglobulin G against cytochrome b5 did not inhibit the alpha-hydroxylase activity. 3. The alpha-hydroxylation activity was more sensitive to trypsin treatment than was NADPH-cytochrome c reductase in rat brain microsomes. This indicates that either alpha-hydroxylase itself or an unknown factor essential in alpha-hydroxylation is highly exposed to the surface of brain microsomes.
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PMID:Alpha-hydroxylation of lignoceroyl-CoA in rat brain microsomes: involvement of NADPH-cytochrome c reductase and topical distribution. 212 39

Transverse-plane topography of mitochondrial outer-membrane long-chain acyl-CoA synthetase was investigated using proteases as probes for exposure of crucial domains, i.e. domains containing the active site or otherwise required for enzymatic activity. Incubation of intact mitochondria with the nonspecific proteases proteinase K and subtilisin resulted in a time-dependent loss of 90% or more of the long-chain acyl-CoA synthetase activity compared to control incubations. The integrity of the outer membrane before and during this treatment was shown by cytochrome c oxidase latency as well as the stability of adenylate kinase activity in the presence of protease. After a 15-min incubation in these conditions, site-specific proteases such as trypsin and chymotrypsin had only a limited inhibitory effect (29 and 58% loss of activity, respectively); however, treatment of hypotonically disrupted mitochondria with these proteases resulted in increased (71 and 77%, respectively) loss of activity. Exposure of trypsin-sensitive crucial domains on the inner surface of the membrane was directly demonstrated by incubation of trypsin-loaded outer-membrane vesicles. Together, these results suggest that mitochondrial long-chain acyl-CoA synthetase is a transmembrane enzyme, possessing crucial domains on both sides of the outer membrane. However, the cytosolic exposure of the enzyme does not appear to be affected by a change in the medium ionic strength as seen previously for other outer-membrane enzymes. In an experiment investigating the topography of the active site of the enzyme, an immobilized substrate analog, desulfo-CoA-agarose, was preincubated with intact mitochondria. This resulted in up to a 42% loss of the activity of long-chain acyl-CoA synthetase, consistent with a cytosolic exposure for at least the CoA-binding domain of the active site.
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PMID:Transverse-plane topography of long-chain acyl-CoA synthetase in the mitochondrial outer membrane. 218 22

Acyl-CoA esters containing the photoreactive acids 12-(4'-azido-2'-nitrophenoxy)[1-14C]dodecanoic acid ([14C]AND-acid) or N-(4'-azido-2'-nitro-[3'-5'-3H]phenyl)-12-aminododecanoic acid ([3H]NANPA-acid) were synthesized. The photoreactive acyl-CoA esters could be bound to bovine acyl-CoA-binding protein (ACBP) and photocrosslinked to the protein. The photocrosslinked acyl-CoA-ACBP complex was separated from unlabelled ACBP on reverse-phase h.p.l.c. and the purified complex was digested with trypsin, Staphylococcus aureus V8 proteinase or endoproteinase Asp-N. By four independent peptide maps it was shown that the amino acids taking part in forming the hydrophobic binding site for acyl-CoA esters in bovine ACBP are located on the peptide segment from Asp21 to Asp38. Both photoreactive acyl-CoA esters used in this study labelled strongly in the segment from Tyr28 to Ala34. 12-(4'-Azido-2'-nitrophenoxy)[1-14C]-dodecanoyl-CoA ([14C]AND-CoA) also introduced a label at position Asp38, but o labelling was found before Ser29. In contrast, N-(4'-azido-2'-nitro[3',5'-3H]phenyl)-12-aminododecanoyl-CoA [3H]NANPA-CoA) also labelled the segment from Asp21 to Tyr28. The difference in labelling by the two photoreactive ligands is most likely caused by different mobility of the arylazido group when linked to the fatty acid either through a phenolic O- or an anilinic N- bond.
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PMID:Determination by photoaffinity labelling of the hydrophobic part of the binding site for acyl-CoA esters on acyl-CoA-binding protein from bovine liver. 222 14

ATP:citrate lyase was purified from the oleaginous yeast Rhodotorula gracilis to homogeneity as judged by polyacrylamide gel electrophoresis, using a novel citrate-Sepharose procedure. The enzyme was found to have a molecular weight of 520,000 and consisted of four identical subunits (Mr = 120,000). Two minor low molecular weight bands were observed on SDS-PAGE (Mr 51,000 and 49,000). Trypsin digestion experiments indicated that these could have been the result of limited proteolysis by an endogenous trypsin-like proteinase. In this respect, it resembles the mammalian ATP:citrate lyase. The enzyme was stimulated by NH+4 ions and inhibited by palmitoyl, lauroyl, oleoyl, myristoyl and stearoyl-CoA esters, glutamate and glucose 6-phosphate but not by acetyl-CoA or shorter chain fatty acyl-CoA esters. The enzyme exhibited normal Michaelis-Menten kinetics for citrate; however there was a 3-fold increase in Km with a high concentration of Cl- ions (0.25 M). The possible regulatory roles of ATP:citrate lyase in R. gracilis are discussed in the light of these findings.
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PMID:ATP:citrate lyase of Rhodotorula gracilis: purification and properties. 230 11

Studies reported from this laboratory have demonstrated that O-glycosidic glycoproteins of salivary, pulmonary, and gastrointestinal origin are acylated by fatty acyltransferase residing in Golgi and microsome-enriched fraction (Slomiany, A., Liau, Y.H., Takagi, A., Laszewicz, W., and Slomiany, B.L. (1984) J. Biol. Chem. 259, 13304-13308). Here we report on the successful purification of this enzyme from rough microsomal membranes of rat gastric mucosa and its identification in a number of diverse tissues and organs, such as heart, liver, pancreas, lung, kidney, salivary glands, and lymphoblasts. The enzymatic activity has been released from the stripped and salt-extracted microsomes with 0.5% Triton X-100 and recovered from 100,000 x g supernatant by affinity chromatography on Cibacron blue F3GA column. The retained fatty acyltransferase protein was selectively displaced from the column with 50 microM palmitoyl-CoA. On nonreducing polyacrylamide gel electrophoresis, the enzymatic activity was associated with a 234-kDa complex, and on sodium dodecyl sulfate polyacrylamide gel electrophoresis, the complex afforded 65- and 67-kDa protein bands. Incubation of microsomes with trypsin prior to enzyme extraction resulted in a 50% inactivation of the fatty acyltransferase and generation of 53- and 55-kDa protein bands, which also had affinity to Cibacron blue F3GA and were displaced from the column together with the active (intact) enzyme. We suggest that the fatty acyltransferase is an integral rough microsomal protein partially exposed to cytosol, which catalyzes the fatty acyl-CoA-protein reaction on the cytosolic site of the rough endoplasmic reticulum and that this enzyme is responsible for processing of the group of protein which are entering rough endoplasmic reticulum-Golgi secretory pathway.
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PMID:Purification of protein fatty acyltransferase and determination of its distribution and topology. 231 87

We found that peroxisomal lignoceroyl-CoA ligase, like palmitoyl-CoA ligase, is present in the peroxisomal membrane whereas the peroxisomal beta-oxidation enzyme system is localized in the matrix. To further define the role of peroxisomal acyl-CoA ligases (membrane component) in providing acyl-CoA for peroxisomal beta-oxidation, we examined the transverse topographical localization of enzymatic sites of palmitoyl-CoA and lignoceroyl-CoA ligases in the peroxisomal membranes. The disruption of peroxisomes by various techniques resulted in the release of a "latent" pool of lignoceroyl-CoA ligase activity while palmitoyl-CoA ligase activity remained the same. Proteolytic enzyme treatment inhibited palmitoyl-CoA ligase activity in intact peroxisomes but had no effect on lignoceroyl-CoA ligase activity. Lignoceroyl-CoA ligase activity was inhibited only if peroxisomes were disrupted with detergent before trypsin treatment. Antibodies to palmitoyl-CoA ligase and to peroxisomal membrane proteins (PMP) inhibited palmitoyl-CoA ligase in intact peroxisomes, and no pool of "latent" activity appeared when antibody-treated peroxisomes were disrupted with detergent. On the other hand, disruption of PMP antibody-treated peroxisomes with detergent resulted in the appearance of a "latent" pool of lignoceroyl-CoA ligase activity. These results demonstrate that the enzymatic site of palmitoyl-CoA ligase is on the cytoplasmic surface whereas that for lignoceroyl-CoA ligase is on the luminal surface of peroxisomal membranes. This implies that palmitoyl-CoA is synthesized on the cytoplasmic surface and is then transferred to the matrix through the peroxisomal membrane for beta-oxidation in the matrix.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Topographical localization of peroxisomal acyl-CoA ligases: differential localization of palmitoyl-CoA and lignoceroyl-CoA ligases. 235 70


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