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)

A peptide containing the globular region of the histone H1 from the fruit fly Ceratitis capitata has been isolated after limited tryptic digestion of insect H1. The composition of this trypsin-resistant core resembles that of the homologous peptide from calf thymus H1, although the insect H1 core possesses one cysteine, two tyrosines, one histidine, and more isoleucine and less glycine and leucine than the calf thymus H1 core. Circular dichroism measurements indicate that all the fragments that possess an ordered secondary structure (approximately 11% in both calf thymus H1 and Ceratitis H1) are present in the trypsin-resistant cores. Both calf thymus and Ceratitis H1 and their trypsin-resistant cores fold cooperatively on titration with NaOH, though the folding of the cores is less cooperative than that for the parentmolecules. On the other hand, salt-induced folding of both cores and intact molecules is noncooperative. The environment of the tyrosyl residues in both calf thymus and Ceratitis H1 has been studied by circular dichroism in the region 250-300 nm and by difference spectroscopy; their pKa' values have also been determined. The results suggest that one of the tyrosyl residues of Ceratitis H1 is buried in the hydrophobic core, in an environment similar to that of calf thymus tyrosine-72, while the second tyrosyl residue of the insect H1 molecule, which titrates with a lower pKa' value (approximately 9.30 in the absence of salt and approximately 9.80 in the presence of 0.3 M KF), is on the surface of the trypsin-resistant core. Due to the limited number of aromatic residues in the histone molecules, the above-mentioned techniques proved to be useful tools to study conformational transitions.
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PMID:Structural studies on histones H1. Circular dichroism and difference spectroscopy of the histones H1 and their trypsin-resistant cores from calf thymus and from the fruit fly Ceratitis capitata. 719 Aug 38

Colipase exists in pancreatic juice in a pro-form which is activated by limited trypsin hydrolysis. During this activation, the N-terminal pentapeptide 1Val-Pro-Asp-Pro-5Arg is cleaved. The new N-terminal sequence formed, 6Gly-Ile-Ile-Ile-10Asn, contains three isoleucine residues. The importance of these for stimulating lipase activity has been investigated by successive Edman degradation of epsilon-acetimidolysine residues followed by limited trypsin hydrolysis. The epsilon-amidinated colipase obtained was fully active both with a phospholipid-covered triacylglycerol (Intralipid) and tributyrin as substrate. After removal of the three isoleucine residues, the activity of colipase was lost with Intralipid but not with tributyrin as substrate. The shortened colipases regained their Intralipid activity upon addition of long-chain fatty acids. The binding of colipase to lipase was not affected by removal of the isoleucine residues.
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PMID:Importance of the N-terminal sequence in porcine pancreatic colipase. 728 23

Two cytochrome P-450 preparations, a constitutive isozyme, form 3b, and a phenobarbital-induced isozyme, form 2, were isolated from rabbit liver microsomes and compared by peptide mapping following digestion with trypsin and by partial sequence analysis. The NH2-terminal sequence of form 3b differed from form 2 in 15 out of 18 amino acids, but both forms have an NH2-terminal methionine residue followed by an acidic residue. Comparisons of many of the tryptic peptides of the two forms by means of high pressure liquid chromatography, as well as amino acid composition and sequence analysis, indicated that peptides from these forms, with one exception, are different. A tridecapeptide, differing only in a methionine (form 3b)/isoleucine (form 2) replacement was isolated from both forms. The amino acid sequence of this peptide is as follows: Met-Pro-Tyr-Thr-Asp-Ala-Val-Ile/Met-His-Glu-Ile-Gln-Arg. Taken together, these data indicate that forms 2 and 3 represent dissimilar gene products. The observation that these two cytochromes share an analogous peptide suggests that this tridecapeptide may contribute structural information necessary for common functional properties.
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PMID:Amino acid sequence of an analogous peptide from two forms of cytochrome P-450. 729 9

During isolation and purification involving fractionation by acetone, ethanol and affinity chromatography on trypsin-Sepharose and isoelectrofocusing hirudin from whole leeches behaves as the inhibitor isolated from leech heads and "pseudohirudin" from leech bodies. The antithrombin activity of hirudin fractions obtained by isoelectrofocusing is about three times less than that of the corresponding hirudin fractions from the heads. The "pseudohirudin" fractions are practically devoid of the antithrombin activity. In hirudin prepared from whole leeches isoleucine and valine were identified as N-terminal amino acids. The antithrombin activity and N-terminal amino acids suggest that hirudin from whole leeches contains admixtures of inactive "pseudohirudin". Hirudin from leech heads purified by complexing with thrombin possesses the activity of 15600 ATNIH units per pg of protein and has isoleucine as an N-terminal amino acid.
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PMID:[Comparative properties of hirudin from whole leeches and from leech heads and bodies]. 738 67

Stereospecific assignments are made for gamma- and delta-methylene hydrogens in a protein by means of estimation of vicinal 1H-1H spin-spin coupling constants from a short-mixing-time TOCSY experiment. 3J alpha beta coupling constants, as measured from a P.E. COSY map, are shown to be well correlated with alpha-beta cross-peak intensities of a short-mixing-time (10 ms) TOCSY map. The procedure is illustrated by application to a trypsin-inhibitor protein (M(r) approximately 7 Kd). Thus, gamma-methylene hydrogens of isoleucine residues have been stereospecifically assigned on the basis of 3J beta gamma 1H-1H coupling patterns and intraresidue cross-peak intensities in a NOESY map; gamma-hydrogens of other residues, such as lysine and arginine, have been stereospecifically assigned solely on the basis of cross-peak intensity patterns resulting from coupling of two beta-hydrogens to two gamma-hydrogens, and in conjunction with stereospecific assignments of beta-methylene hydrogens. However, intraresidue NOE intensities are needed if one or two pairs of coupling constants cannot be estimated because of cross peaks either overlapping or occurring proximal to the diagonal. The delta-methylene hydrogens have been stereospecifically assigned on the basis of coupling between two gamma-hydrogens and two delta-hydrogens, in combination with stereospecific assignments of gamma-hydrogens. Stereospecific assignments of side chains should contribute to the overall precision and accuracy of NMR-determined three-dimensional solution structures of proteins.
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PMID:A practical method for stereospecific assignments of gamma- and delta-methylene hydrogens via estimation of vicinal 1H-1H coupling constants. 759 51

A new sulfated, cyclic depsipeptide, called cyanopeptolin S, from Microcystis sp. was isolated from a water bloom in the Auensee/Leipzig (Germany). The depsipeptide had a relative molecular mass of 925 and contained L-arginine, L-threonine, L-isoleucine, N-methyl-L-phenylalanine, a L-glutamic acid-delta-aldehyde ring system and a sulfated D-configurated glyceric acid as a side chain. The structure was elucidated by means of two-dimensional 1H and 13C nuclear magnetic resonance spectroscopy, fast atom bombardment mass spectroscopy. Fourier transformed infrared spectroscopy and combined gas-liquid chromatography/mass spectrometry. Cyanopeptolin S inhibited trypsin with an IC50 < or = 0.2 micrograms ml-1.
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PMID:Cyanopeptolin S, a sulfate-containing depsipeptide from a water bloom of Microcystis sp. 760 93

Because of the conflicting conclusions that have been reached regarding the location of the two putative membrane-spanning segments from cysteine 911 through isoleucine 929 and from isoleucine 946 through cysteine 964 in the alpha subunit of native ovine Na+/K(+)-transporting ATPase, the disposition of lysine 943 with respect to the plane of the lipid bilayer was investigated. Sealed, right-side-out vesicles were modified with pyridoxal phosphate and Na[3H]BH4 in the presence and absence of saponin, a reagent that creates holes in the membranes. Modified alpha polypeptide was isolated, and digested with trypsin and chymotrypsin to release the desired peptides, QQGMK and QQGMK([3H]pyr)NK (where [3H]pyr designates the modification on lysine 943). These peptides, after cyclization of their amino-terminal glutamines, were isolated with an immunoadsorbent specific for the amino-terminal sequence pyroglutamyl-QGM-followed by high-pressure liquid chromatography on a C-18 reverse phase column. Comparisons were made of the extent of incorporation of radioactivity into lysine 943 between sealed vesicles and sealed vesicles pretreated with saponin. An increase in incorporation into lysine 943 of 5-fold to 18-fold was seen in vesicles pretreated with saponin prior to the modification with pyridoxal phosphate. This increase in incorporation is consistent with a cytoplasmic location for lysine 943. This conclusion places the residues on the carboxy-terminal side of the putative membrane-spanning segment from cysteine 911 through isoleucine 929 and the amino-terminal side of the putative membrane-spanning segment from isoleucine 946 through cysteine 964 in the ovine alpha subunit on the cytoplasmic side of the membrane.
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PMID:Topological disposition of lysine 943 in native Na+/K(+)-transporting ATPase. 762 20

Porcine colipase, the protein cofactor of pancreatic lipase, was isolated from pancreas freshly collected on animals and from a side fraction from the production of insulin (Novo Nordisk A/S). Samples of purified colipase were analyzed for homogeneity by polyacrylamide gel electrophoresis, reverse-phase high-performance liquid chromatography (RPLC), quantitative N-terminal sequence determination and mass spectrometry. The activating properties of colipase preparations were assayed against tributyrin, triolein or the commercial Intralipid emulsion, in presence of bile salt. Two fractions of colipase with the same specific activity were purified from fresh pancreas. The major fraction (85%) contained one single protein corresponding to fragment 1-93 of the 95-residue form of colipase (procolipase) previously characterized in porcine pancreatic juice. The other fraction (15%) corresponded to fragment 1-91 of procolipase. Also, two fractions of colipase were purified from the side fraction supplied by Novo. These fractions consisted of the 95-residue proform of colipase and of fragment 1-93, respectively, both specifically cleaved at the Ile79-Thr80 peptide bond with partial removal of isoleucine at position 79 and serine at position 78. Procolipase split at the 79-80 bond retained full activity on tributyrin and triolein and on the Intralipid emulsion but the kinetics of hydrolysis of triacylglycerol substrates showed much longer lag periods than those observed with native procolipase. Also, all forms of procolipase split at the 79-80 bond showed one peak in RPLC but their retention time was markedly decreased as compared to that of native procolipase which indicated a weaker hydrophobic binding capacity. The value of the retention time was of the same order of magnitude as that of inactive reduced procolipase. Treatment of native procolipase by pancreatic endopeptidases showed that elastase is likely responsible for specific cleavage at the 79-80 bond of procolipase purified from the Novo extract. Limited proteolysis by trypsin of the proforms of colipase split at the 79-80 bond reduced the lag period. Results presented in this communication provide the first direct evidence showing that the finger-shaped peptide segment between half-cystine residues at positions 69 and 87 is involved in colipase-lipid interaction as previously hypothesized from the three-dimensional structure of the protein.
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PMID:Lipid binding and activating properties of porcine pancreatic colipase split at the Ile79-Thr80 bond. 769 7

The human immunodeficiency virus, type 1 (HIV-1) genome encodes a 15-kDa accessory gene product, Vpr, that is essential for virus replication in primary monocytes/macrophages. Being present in the virion, Vpr is believed to function in the early phases of HIV-1 replication, including nuclear migration of the pre-integration complex and/or transcription of the provirus genome. By gel filtration analysis of highly purified Vpr protein and its mutants, we demonstrate that HIV-1 Vpr exists as an oligomer. The N-terminal domain of Vpr (amino acids (aa) 1-42) is sufficient for oligomerization; however, deletion of aa 36-76 from Vpr disrupts oligomerization, suggesting that aa 36-42 are critical for Vpr oligomerization. As a result of Vpr oligomerization, basic aa residues within Vpr aa 1-73 are highly resistant to trypsin digestion, while those within Vpr aa 74-96 are easily accessible. Mutations within the leucine-/isoleucine-rich domain (aa 60-81), which was previously identified to be involved in Vpr interaction with a host cellular protein, rendered Arg62 more susceptible to trypsin digestion. Thus, the Vpr oligomeric structure must be extended into this domain. These results suggest a novel feature of HIV-1 Vpr that may be important for its functions.
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PMID:Biochemical mechanism of HIV-1 Vpr function. Oligomerization mediated by the N-terminal domain. 779 8

In addition to the known 94-kd gelatinase (matrix metalloproteinase 9, MMP-9), HL-60 leukemia cells release a hither-to undescribed 45-kd metalloproteinase into the culture medium. This enzyme cleaves the synthetic substrate Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg, which represents the cleavage site for collagenases in collagen type I not between isoleucine and alanine--the typical cleavage site for collagenases--but between alanine and glycine. The enzymatic activity was purified through a combination of zinc-chelate-Sepharose column chromatography, precipitation with Fractogel TSK-AF Red and gelatin-Sepharose, and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Microsequence analysis of the NH2-terminus of the purified 45-kd proteinase revealed the sequence Asp-Ile-Ser-Lys-Tyr-Thr-Thr-Thr-, which could not be found in other proteins when searched in several protein data bases. Incubation of the enzyme immobilized on nitrocellulose membranes with polyclonal antibodies to collagenase and stromelysin or gelatinases revealed no cross-reactivity. The proteolytic activity was not increased by treatment with trypsin, 8M urea, acid, or organomercurials. The proteinase, which was inhibited by chemical inhibitors of metalloproteinases, such as phenanthrolene or EDTA, is able to degrade several matrix constituents, such as collagen type IV, fibronectin, gelatin, and proteoglycans. In contrast to all known MMPs, the proteolytic activity of the 45-kd enzyme was not abolished upon incubation with recombinant tissue inhibitors of matrix metalloproteinases (TIMP) 1 or 2. Thus, the novel enzyme may influence extracellular matrix (ECM) turnover in vivo because its activity is not influenced by specific inhibitors of MMPs.
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PMID:Leukemic cells (HL-60) produce a novel extracellular matrix-degrading proteinase that is not inhibited by tissue inhibitors of matrix metalloproteinases (TIMPs). 782 72


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