Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mitochondrial DNA-binding protein P16 was isolated from rat liver mitochondrial lysates by affinity chromatography on single strand DNA agarose and separated from DNA in the preparation by alkaline CsCl isopycnic gradients. The top fraction of the gradients contained a single polypeptide species (Mr approximately equal to 15,200) based upon SDS PAGE. Digestion of single strand DNA-bound P16 with proteinase K produced a protease-insensitive, DNA-binding fragment (Mr approximately equal to 6,000) that has been purified by essentially the same procedures used for intact P16. The partial amino acid compositions for P16 and the DNA-binding fragment were obtained by conventional methods. Analysis of subcellular fractions revealed that nearly all of the cellular P16 was located in the mitochondria and that only trace amounts of protein of comparable electrophoretic mobility could be isolated from the nuclear or cytoplasmic fractions. The labeling of P16 with [35S]methionine in primary rat hepatocyte cultures was inhibited by more than 90% by the cytoplasmic translation inhibitor cycloheximide, but unaffected by the mitochondrial-specific agent chloramphenicol. These results indicate that P16 is synthesized on cytoplasmic ribosomes and imported into the mitochondria. The addition of purified P16 to deproteinized mitochondrial DNA resulted in the complete protection of the labeled nascent strands of displacement loops against branch migrational loss during cleavage of parental DNA with SstI, thus providing strong evidence that P16 is the single entity required for this in vitro function. Incubation of P16 with single strand phi X174 DNA, double strand (RF) phi X174 DNA, or Escherichia coli ribosomal RNA and subsequent analysis of the nucleic acid species for bound protein indicated a strong preference of P16 for single strand DNA and no detectable affinity for RNA or double strand DNA. Examination of P16-single strand phi X174 DNA complexes by direct electron microscopy revealed thickened, irregular fibers characteristic of protein-associated single strand DNA.
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PMID:Purification and general properties of the DNA-binding protein (P16) from rat liver mitochondria. 403

The lipopolysaccharide (LPS) of Neisseria gonorrhoeae whole-cell lysates and proteinase K-digested lysates was examined and compared with purified homologous LPS by a method which preferentially stains LPS in polyacrylamide gels. The silver-stained profile of gonococcal LPS in the proteinase K-digested lysate was similar to that of homologous purified LPS; however, the LPS profile in whole-cell lysates was much smaller than that of digested lysates or purified LPS. Conditions of solubilization did not affect these differences. Since it is known that LPS migrates in a unique fashion in second-dimension electrophoresis, the location of LPS in the whole-cell lysates was probed by second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a variety of stains and radiolabels. Results from these experiments indicated a stable and reproducible association of LPS with proteins ranging between 23,000 to 36,000 in Mr, in particular major outer membrane protein I. In addition to staining with the silver method, which preferentially stains LPS, the putative LPS was resistant to digestion by proteinase K, did not stain with Coomassie brilliant blue, and was not labeled extrinsically with 125I (Iodogen method) or intrinsically with [35S]methionine. Analysis of two-dimensional gels by immunoblotting with rabbit antisera prepared from protein I bands removed from a polyacrylamide gel revealed the presence of antigens in the same area of the gel (below proteins that were 23,000 to 36,000 in Mr). Antibodies to constituents which migrated below the diagonal were essentially removed by adsorption of antisera with purified LPS, as were antibodies to homologous LPS and LPS in proteinase K-digested whole-cell lysates. Immunoblotting with a monoclonal antibody specific for LPS demonstrated reactivity of the antibody with LPS and with the protein I band. On the basis of these data, we conclude that protein I and perhaps other proteins in the whole-cell lysate are stably associated with LPS; this complex is resistant to dissociation in sodium dodecyl sulfate at high temperature (approximately 100 degrees C) but does, for unknown reasons, dissociate with electrophoresis in the second dimension. The association of LPS with protein antigens in sodium dodecyl sulfate-polyacrylamide gels adds another dimension of complexity to analysis of these antigens by immunoelectroblotting. Furthermore, the tight association of LPS with the major outer membrane protein I may alter the nature of the immune response generated by "purified" protein I vaccine antigens. The possible role of protein-LPS complexes in the pathogenesis of gonorrhea is discussed.
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PMID:Analyses of gonococcal lipopolysaccharide in whole-cell lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: stable association of lipopolysaccharide with the major outer membrane protein (protein I) of Neisseria gonorrhoeae. 620 9

Attachment of [35S]methionine-labelled mammalian type 3 reovirus to murine L cells and human HeLa cells was studied under equilibrium conditions. Cellular attachment sites could be completely saturated with 35S-labelled reovirus, indicating that specific attachment sites for reovirus are present on the surface of these cells. We calculated that L cells possess about 86000-105000 attachment sites per cell while HeLa cells possess about 126000-147000 sites per cell for type 3 reovirus. Unlabelled reovirus was highly efficient in competing for attachment by 35S-labelled reovirus to the saturable attachment sites of both L and HeLa cells, further indicating the specificity of the interaction. We also found that unlabelled reovirus competed equally well for both binding and internalization of 35S-labelled reovirus into murine L cells, suggesting that the L cell attachment site may serve as a virus entry site. Phospholipase digestion of L cells had no effect on subsequent reovirus attachment, while treatment of L cells with moderate concentrations of bromelain (but not trypsin, proteinase K or pronase) and Vibrio cholerae neuraminidase reproducibly decreased subsequent reovirus attachment. These results and those of others (Epstein et al., 1984, Virology 133, 46-55) suggest that mammalian reoviruses attach to specific cell surface receptors on at least two species of mammalian cells to initiate the infectious cycle.
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PMID:Saturable attachment sites for type 3 mammalian reovirus on murine L cells and human HeLa cells. 639 64

Proteolysis of glucose dehydrogenase from Bacillus megaterium with proteinase K apparently generated two fragments. The small fragment, designated as K-peptide, was sequenced and its covalent structure was determined as Ser-Ser-Glu-Ala-Ser-Tyr-Val-Thr-Gly-Ile-Thr-Leu-Phe-Ala-Asp-Gly-Gly-Met-Thr- Gln-Tyr-Pro-Ser-Phe-Glu-Ala-Gly-Arg-Gly. The sequence analysis showed that the K-peptide consists of two identical fragments, one of which is lacking one serine residue at the amino-terminus.
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PMID:Amino acid sequence of the K-peptide generated by limited proteolysis of glucose dehydrogenase from Bacillus megaterium by proteinase K1. 642 50

Fatal familial insomnia (FFI) and a subtype of familial Creutzfeldt-Jakob disease (CJD178) are two prion diseases that have different clinical and pathological features, the same aspartic acid to asparagine mutation (D178N) at codon 178 of the prion protein (PrP) gene, but distinct genotypes generated by the methionine-valine polymorphism at codon 129 (129M or 129V) in the mutant allele of the PrP gene. The D178N, 129M allele segregates with FFI while the D178N, 129V allele segregates with CJD178. The proteinase K resistant PrP (PrPres) isoforms present in FFI and CJD178 differ in degree of glycosylation and size. Thus, the amino acid, methionine or valine, at position 129 of the mutant allele, in conjunction with D178N mutation results in significant alterations of PrPres in FFI and CJD178. The 129 polymorphic site also exerts influence through the normal allele: the course of the disease is shorter in the patients homozygous at codon 129 and other minor but consistent phenotypic differences occur between homozygous and heterozygous FFI patients. The comparative study of PrPres distribution in FFI homozygotes and heterozygotes at codon 129 has lead to the conclusion that the phenotypic differences observed between these two FFI patient populations may be the result of different rates of conversion of normal PrP into PrPres, at least in some brain regions.
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PMID:Fatal familial insomnia and familial Creutzfeldt-Jakob disease: clinical, pathological and molecular features. 776 90

Proteins or peptides having an N-terminal-blocked amino acid were successively digested by pronase E, proteinase K, and carboxypeptidase Y. The N-blocked amino acids released from proteins or peptides were derivatized with 9-anthryldiazomethane (ADAM) to the corresponding esters followed by addition of formic acid to remove the remaining ADAM which interfered with further analysis. The anthryl esters were analyzed by high-performance liquid chromatography equipped with a fluorimetric detector. Twelve N-acetylamino acids (Asn, Gln, Ser, Thr, Gly, Ala, Tyr, Pro, Met, Val, Ile, and Leu), pyroglutamic acid, N-formylMet, and N-myristoylGly could be separated from each other and identified on the same chromatographic run. As examples of applied experiments to proteins and peptides, N-acetyl derivatives of Ser, Ala, Met, Gly, Tyr, and Pro as well as N-myristoylGly could be satisfactorily identified using 100 pmol each of seven proteins and peptides. The method reported here is an improved one that was reported in the previous paper based on the same principles.
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PMID:Microidentification of N-terminal-blocked amino acid residues of proteins and peptides. 797 59

We incubated yeast cells (Saccharomyces cerevisiae) with the methyl donor S-adenosyl-L-[methyl-3H]methionine and then fractionated their cellular components by gel electrophoresis in sodium dodecyl sulfate. By analyzing gel slices for [3H]methyl esters by a vapor-phase diffusion assay, we detect major methyl-esterified species that migrate at apparent polypeptide sizes of 24 and 22 kDa and minor species of 49, 38, 35, 33, 31, and 26 kDa. Incubation of extracts from labeled cells with ribonuclease A or proteinase K revealed that the 24- and 22-kDa species represent methyl-esterified RNAs, whereas the other species are methyl-esterified polypeptides. The 38-, 33-, 31-, and 26-kDa polypeptides were not methyl-esterified in an isogenic yeast strain lacking the STE14 gene encoding a C-terminal isoprenylcysteine methyltransferase, suggesting that they are substrates for the STE14 methyltransferase. On the other hand, the amount of the methylated 49-kDa polypeptide is reduced in the ste14 mutant, indicating that at least two methylated polypeptides are present--one a substrate of the STE14 methyltransferase and one a substrate of a STE14-independent methyltransferase. The 35-kDa polypeptide also appears to be methylated by a STE14-independent methyltransferase. When cells were incubated in the presence of the protein synthesis inhibitor cycloheximide, little or no methylation of the STE14-dependent species was detected while the methylation of the STE14-independent substrates was unaffected. Pulse-chase studies revealed significant turnover of all of the methylated species in a 4-h period, with the exception of the 38-kDa polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Protein carboxyl methylation in Saccharomyces cerevisiae: evidence for STE14-dependent and STE14-independent pathways. 806 61

Tegumental extracts from adult worms of Schistosoma mansoni contain an inhibitory activity to the S. mansoni 28-kDa serine protease and to pancreatic elastase. By using biotinylated elastase and streptavidin-agarose, the postulated protease inhibitor has been isolated from the crude worm extract in a single step. Monospecific rabbit antibodies raised against the protease inhibitor have immunoprecipitated a 56-kDa [35S]Met-labeled serine protease inhibitor which was designated Smpi56 (S. mansoni protease inhibitor, 56 kDa). Smpi56 binds tightly to and inhibits the 28-kDa protease of S. mansoni and pancreatic and neutrophil elastase but not papain, pepsin, thrombin, trypsin, chymotrypsin, proteinase K, urokinase and acetylcholinesterase. The biological function of Smpi56 is still not known, but in view of its elastase inhibitory activity it may be speculated that the parasite is employing Smpi56 to protect itself from activated neutrophils. Smpi56 may also potentially protect the parasite from its endogenous 28-kDa protease.
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PMID:Schistosoma mansoni: isolation and characterization of Smpi56, a novel serine protease inhibitor. 811 69

Sm23, a surface protein of the human parasite Schistosoma mansoni, belongs to the family of "cysteine-rich, hydrophobic proteins," which are expressed on mammalian hematopoietic cells or tumor cells. Sm23 shares the highly conserved hydrophobicity profile of these proteins, which predicts four transmembrane segments, but is in addition linked to the membrane by a glycosylphosphatidylinositol (GPI) anchor. Our results suggest that Sm23 uses both the potential transmembrane domains and the GPI anchor for membrane insertion: (a) Sm23 was not released from the surface after cleavage with phosphatidylinositol-specific phospholipase C (PIPLC). (b) In a Triton X-114 phase-separation system, native [3H]ethanolamine- or [35S]methionine-labeled Sm23 partitioned into the detergent phase. Upon removal of the GPI anchor by PIPLC, the majority of the molecules stayed in the detergent-phase as expected of a transmembrane protein. (c) When full-length recombinant Sm23 was transcribed and translated in vitro, the polypeptide chain was inserted into microsomal membranes: Sm23 stayed associated with the membranes when they were incubated with carbonate buffer at pH 11.5, and membrane bound Sm23 was protected from digestion with proteinase K. (d) Recombinant Sm23, when expressed in the baculovirus expression system, was transported to the surface of infected insect cells, and similarly to the native protein it was not released from these cells after cleavage with PIPLC.
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PMID:Schistosoma mansoni: Sm23 is a transmembrane protein that also contains a glycosylphosphatidylinositol anchor. 816 Nov 93

In the association of serine proteinases with their cognate substrates and inhibitors an important interaction is the fitting of the P1 side chain of the substrate or inhibitor into a preformed cavity of the enzyme called the S1 pocket. In turkey ovomucoid third domain, which is a canonical protein proteinase inhibitor, the P1 residue is Leu18. Here we report the values of equilibrium constants, Ka, for turkey ovomucoid third domain and 13 additional Leu18X variants with six serine proteinases: bovine alpha chymotrypsin A, porcine pancreatic elastase, subtilisin Carlsberg, Streptomyces griseus proteinases A and B, and human leukocyte elastase. Eight of the Xs are coded amino acids: Ala, Ser, Val, Met, Gln, Glu, Lys, and Phe, and five are noncoded: Abu, Ape, Ahx, Ahp, and Hse. They were chosen to simplify the interamino acid comparisons. In the homologous series of straight-chain side chains Ala, Abu, Ape, Ahx, Ahp, free energy of binding decreases monotonically with the side-chain length for chymotrypsin with large binding pocket, but even for this enzyme shows curvature. For the two S. griseus enzymes a minimum appears to be reached at Ahp. A minimum is clearly evident for the two elastases, where increasing the side-chain length from Ahx to Ahp greatly weakens binding, but much more so for the apparently more rigid pancreatic enzyme than for the more flexible leukocyte enzyme. beta-Branching (Ape/Val) is very deleterious for five of the six enzymes; it is only slightly deleterious for the more flexible human leukocyte elastase. The effect of gamma-branching (Ahx/Leu), of introduction of heteroatoms (Abu/Ser), (Ape/Hse), and (Ahx/Met), and of introduction of charge (Gln/Glu) and (Ahp/Lys) are tabulated and discussed. An important component of the free energy of interaction is the distortion of the binding pocket by bulky or branched side chains. Most of the variants studied were obtained by enzymatic semisynthesis. X18 variants of the 6-18 peptide GlyNH2 were synthesized and combined with natural reduced peptide 19-56. Disulfide bridges were formed. The GlyNH2 was removed and the reactive-site peptide bond X18-Glu19 was synthesized by complex formation with proteinase K. The resultant complexes were dissociated by sudden pH drop. This kinetically controlled dissociation afforded virgin, reactive-site-intact inhibitor variants.
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PMID:Binding of amino acid side chains to preformed cavities: interaction of serine proteinases with turkey ovomucoid third domains with coded and noncoded P1 residues. 849 99


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