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

The topography of membrane-surface-exposed amino acids in the light-driven proton pump bacteriorhodopsin (BR) was studied. By limited proteolysis of purple membrane with papain or proteinase K, domains were cleaved, separated by SDS-PAGE, and electroblotted onto polyvinylidene difluoride (PVDF) membranes. Fragments transferred were sequenced in a gas-phase sequencer. Papain cleavage sites at Gly-65, Gly-72, and Gly-231, previously only deduced from the apparent molecular weight of the digestion fragments, could be confirmed by N-terminal micro-sequencing. By proteinase K, cleavage occurred at Gln-3, Phe-71, Gly-72, Tyr-131, Tyr-133, and Ser-226, i.e., in regions previously suggested to be surface-exposed. Additionally, proteinase-K cleavage sites at Thr-121 and Leu-127 were identified, which are sites predicted to be in the alpha-helical membrane-spanning segment D. Our results, especially that the amino acids Gly-122 to Tyr-133 are protruding into the aqueous environment, place new constraints on the amino-acid folding of BR across the purple membrane. The validity of theoretical prediction methods of the secondary structure and polypeptide folding for membrane proteins is challenged. The results on BR show that micro-sequencing of peptides separated by SDS-PAGE and blotted to PVDF can be successfully applied to the study of membrane proteins.
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PMID:Topography of surface-exposed amino acids in the membrane protein bacteriorhodopsin determined by proteolysis and micro-sequencing. 291 38

A simple procedure is described for the preparation of photosystem I (PSI) particles from Triton X-100-solubilized thylakoid membranes of the unicellular cyanobacterium Synechococcus 6301. The purified PSI complex contained the full complement of antenna chlorophylls, 130 +/- 5/P700, displayed the electron paramagnetic resonance signals characteristic of iron-sulfur centers X, A, and B, and had a protein/chlorophyll ratio of 2.9. Determination of the polypeptide composition, utilizing a uniformly 14C-labeled complex, showed that it contained polypeptides of 70, 18, 17.7, 16, and 10 kDa, in a molar ratio of 4.0:0.7:1.0:0.5:1.6. The relative amount of the lower molecular weight polypeptides showed progressive decrease with increase in Triton X-100 concentration and time of exposure to detergent. Consequently, it is proposed that in vivo the composition of the complex is [70 kDa]4 [18 kDa]1 [17.7 kDa]1 [16 kDa]1 [10 kDa]2. Relative to 130 mol of chlorophyll a, the PSI complex contained 16 mol of carotenoids, 13.7 +/- 1.0 g atoms of Fe, and 12.2 +/- 1.1 g atoms of labile sulfide. The properties of complexes fully depleted of the low-molecular weight polypeptides by treatment with sodium dodecyl sulfate or with proteinase K are also described.
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PMID:Characterization of a cyanobacterial photosystem I complex. 298 Dec 24

The genome of infectious bursal disease virus (IBDV) consists of two segments of double-stranded (ds)RNA with molecular weights of 2.2 X 10(6) and 1.9 X 10(6) Da, respectively. After treatment of IBDV particles with proteinase K in the presence of sodium dodecyl sulfate (SDS), linear dsRNA molecules are released from the virus particles. However, after heating of virus particles at 100 degrees for 3 min in 1.5% SDS, without the protease, dsRNA-protein complexes can be seen under the electron microscope: Knob-like proteinaceous structures are linked to the ends of the dsRNA molecules of either size class which are circularized to form individual rings. A 90,000-Da IBDV structural polypeptide, the only protein encoded by the smaller genome segment, has been demonstrated to remain firmly linked to the IBDV genome under these conditions. No functional data exist about this circularizing protein; it is a probable candidate for an RNA-dependent RNA polymerase or an assembly protein for the two dsRNA segments. At high particle concentrations, or when the preparations are allowed to stand for several hours before spreading, these complexes tend to aggregate to form flower-like structures.
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PMID:The two segments of the infectious bursal disease virus genome are circularized by a 90,000-Da protein. 303 77

BHK cells transfected with human lysosomal acid phosphatase (LAP) cDNA (CT29) expressed 70-fold higher enzyme activities of acid phosphatase than non-transfected BHK cells. The CT29-LAP was synthesized in BHK cells as a heterogeneously glycosylated precursor that was tightly membrane associated. Transfer to the trans-Golgi was associated with a small increase in size (approximately 7 kd) and partial processing of the oligosaccharides to complex type structures. CT29-LAP was transferred into lysosomes as shown by subcellular fractionation, immunofluorescence and immunoelectron microscopy. Lack of mannose-6-phosphate residues suggested that transport does not involve mannose-6-phosphate receptors. Part of the membrane-associated CT29-LAP was processed to a soluble form. The mechanism that converts CT29-LAP into a soluble form was sensitive to NH4Cl, and reduced the size of the polypeptide by 7 kd. In vitro translation of CT29-derived cRNA in the presence of microsomal membranes yielded a CT29-LAP precursor that is protected from proteinase K except for a small peptide of approximately 2 kd. In combination with the sequence data available for LAP, these observations suggest that CT29-LAP is synthesized and transported to lysosomes as a transmembrane protein. In the lysosomes, CT29-LAP is released from the membrane by proteolytic cleavage, which removes a C-terminal peptide including the transmembrane domain and the cytosolic tail of 18 amino acids.
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PMID:Human lysosomal acid phosphatase is transported as a transmembrane protein to lysosomes in transfected baby hamster kidney cells. 305 14

A 12 Kd antigen was isolated from Fasciola hepatica adult worm extracts by gel filtration in Sephadex G-50 and ion exchange chromatography using DEAE Sephadex A-120. Mice immunized with this Fasciola-derived 12 Kd antigen developed antibodies to Schistosoma mansoni adult worm extracts, demonstrating its cross-reactivity with schistosomes. Vaccination of mice with microgram amounts of the antigen in Freund's adjuvant induced up to 77% reduction in worm burdens after challenge with S. mansoni cercariae. F. hepatica 12 Kd degraded by proteinase K to lower molecular weight polypeptides which still retain their antigenicity as determined by the enzyme-linked immunoelectrotransfer blot technique. Treatment with either Endoglycosidase H, neuraminidase, or dithiothreitol had no effect on its mobility in sodium dodecyl sulfate polyacrylamide gels, or in its recognition by antibody, suggesting the absence of carbohydrate moieties or disulphide bonds in relation to its antigenic determinants and also suggesting that the antigen is a pure polypeptide. These studies establish that a molecularly defined cross-reactive component of one parasitic trematode (F. hepatica) induces resistance to challenge infection with another parasitic trematode (S. mansoni). Its polypeptide nature makes recombinant DNA technology an alternative for the manufacture of a vaccine.
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PMID:Successful vaccination against murine Schistosoma mansoni infection with a purified 12 Kd Fasciola hepatica cross-reactive antigen. 312 43

Absorption of serum from chronically infected mice with homogenized schistosome eggs reduced antibody binding to the schistosomulum surface by 94%, indicating that almost all schistosomulum surface recognition during chronic infection is due to epitopes shared with the egg. Absorption of the serum with egg homogenate from which protein antigens had been removed by boiling and digestion with proteinase K resulted in a similar reduction of antisurface antibody demonstrating that all the shared epitopes that are recognized are carbohydrate in nature. Analysis of the time course of anticarbohydrate antibody production and the levels of antibody in mice infected with a single sex of schistosome indicated that eggs directly stimulated this response. Mouse mAb were identified that bound at very high levels to the schistosomulum surface and that recognized carbohydrate epitopes shared with the egg. Three of these had previously been demonstrated to passively transfer resistance, indicating that these surface carbohydrates are potential targets of protective immunity in the mouse. All the anticarbohydrate mAb also bound to the surface of schistosomula of other schistosome species. Thus, the strong immune response against these epitopes in chronic infection could account for the cross-specific immunity observed. Mice vaccinated with irradiated cercariae lacked high levels of anticarbohydrate antibodies and their recognition of the surface was largely due to antibody to species-specific polypeptide epitopes. With respect to the Mr greater than 200,000 and 38,000 antigens, it was demonstrated that these epitopes were present on the same antigens that bear the carbohydrate moieties recognized by antibodies from chronically infected mice. This specific polypeptide recognition is also reflected in the immunity generated by exposure to irradiated cercariae.
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PMID:Analysis of the anti-Schistosoma mansoni surface antibody response during murine infection and its potential contribution to protective immunity. 312 16

We have previously demonstrated that a Fasciola hepatica-derived adult worm antigen, which is cross-reactive with Schistosoma mansoni and designated FhSmIII(M), induces resistance to challenge infection with S. mansoni in mice. The current review concerns the methods developed to isolate and partially characterize a major component of FhSmIII(M), a 12-kDa polypeptide, as well as immunity studies involving this antigen. Utilizing conventional gel filtration, followed by diethylaminoethyl (DEAE) Sephadex A-120 and monitoring the fractions by polyacrylamide gel electrophoresis (PAGE) and enzyme-linked immunoelectrotransfer blot techniques (EITB), we were able to isolate the 12-kDa antigenic polypeptide to homogeneity. Conventional gel filtration chromatography was followed by high-pressure, liquid anion, exchange chromatography, when highly purified material was needed, although the effective yields diminished drastically with the latter. Mice, rabbits and calves with a primary infection of F. hepatica developed antibodies (detectable in enzyme linked immunosorbent assay (ELISA) to the F. hepatica 12-kDa polypeptide within 2 weeks of infection. Mice with a primary infection of S. mansoni developed significant, but low, levels of anti-12-kDa antibodies by 7 weeks post-infection. Immunization of mice with microgram amounts of this 12-kDa polypeptide in Freunds' adjuvant resulted in the development of up to 77% less S. mansoni worms than the controls. Treatment with either endoglycosidase H, neuraminidase or dithiothreitol had no effect on the protein's mobility on sodium dodecyl sulfate (SDS)-PAGE or in its recognition by antibodies, suggesting the absence of carbohydrate moieties or disulphide bonds in relation to its antigenic determinants. Degradation by proteinase K further confirmed its polypeptide nature and points to recombinant DNA technology for the large-scale manufacture of this potential vaccine. Further use of this antigen in immunity studies should greatly contribute to the clarification of the mechanisms involved in cross-resistance against schistosomiasis.
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PMID:Acquired immunity in schistosomiasis with purified Fasciola hepatica cross-reactive antigens. 314 81

The major coat protein of the filamentous bacteriophage M13 is a 50-residue amphiphilic polypeptide which is inserted, as an integral membrane-spanning protein, in the inner membrane of the Escherichia coli host during infection. 13C was incorporated biosynthetically into a total of 23 of the peptide carbonyls using labeled amino acids (alanine, glycine, lysine, phenylalanine, and proline). The structure and dynamics of carbonyl-labeled M13 coat protein were monitored by 13C nuclear magnetic resonance (NMR) spectroscopy. Assignment of many resonances was achieved by using protease digestion, pH titration, or labeling of the peptide bond with both 13C and 15N. The carbonyl region of the natural-abundance 13C NMR spectrum of M13 coat protein in sodium dodecyl sulfate solution shows approximately eight backbone carbonyl resonances with line widths much narrower than the rest. Three of these more mobile residues correspond to assigned peaks (glycine-3, lysine-48, and alanine-49) in the individual amino acid spectra, and another almost certainly arises from glutamic acid-2. A ninth residue, alanine-1, also gives rise to a very narrow carbonyl resonance if the pH is well above or below the pKa of the terminal amino group. These data suggest that only about four residues at either end of the protein experience large-amplitude spatial fluctuations; the rest of the molecule is essentially rigid on the time scale of the overall rotational tumbling of the protein-detergent complex. The relative exposure of different regions of detergent-bound protein was monitored by limited digestion with proteinase K.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Backbone dynamics of a model membrane protein: assignment of the carbonyl carbon 13C NMR resonances in detergent-solubilized M13 coat protein. 330 12

The coding region for the mature form of TEM beta-lactamase was fused to random positions within the coding region of the penicillin-binding protein 1B (PBP 1B) gene and the nucleotide sequences across the fusion junctions of 100 in-frame fusions were determined. All fusion proteins that contained at least the NH2-terminal 94 residues of PBP 1B provided individual cells of E. coli with substantial levels of ampicillin resistance, suggesting that the beta-lactamase moiety had been translocated to the periplasm. Fusion proteins that contained less than or equal to 63 residues of PBP 1B possessed beta-lactamase activity, but could not protect single cells of E. coli from ampicillin, indicating that the beta-lactamase moiety of these fusion proteins remained in the cytoplasm. The beta-lactamase fusion approach suggested a model for the organization of PBP 1B in which the protein is embedded in the cytoplasmic membrane by a single hydrophobic transmembrane segment (residues 64-87), with a short NH2-terminal domain (residues 1-63), and the remainder of the polypeptide (residues 88-844) exposed on the periplasmic side of the cytoplasmic membrane. The proposed model for the organization of PBP 1B was supported by experiments which showed that the protein was completely digested by proteinase K added from the periplasmic side of the cytoplasmic membrane but was only slightly reduced in size by protease attack from the cytoplasmic side of the membrane.
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PMID:Use of a beta-lactamase fusion vector to investigate the organization of penicillin-binding protein 1B in the cytoplasmic membrane of Escherichia coli. 333 Jul 53

The major portion of the eukaryotic genome consists of various categories of repetitive DNA sequences which have been studied with respect to their base compositions, organizations, copy numbers, transcription and species specificities; their biological roles, however, are still unclear. A novel quality of a highly repetitive mouse DNA sequence is described which points to a functional role: All copies (approximately 50,000 per haploid genome) of this DNA sequence reside on genomic Alu I DNA fragments each associated with nuclear polypeptides that are not released from DNA by proteinase K, SDS and phenol extraction. By this quality the repetitive DNA sequence is classified as a member of the sub-set of DNA sequences involved in tight DNA-polypeptide complexes which have been previously shown to be components of the subnuclear structure termed 'nuclear matrix'. From these results it has to be concluded that the repetitive DNA sequence characterized in this report represents or comprises a signal for a large number of site specific attachment points of the mouse genome in the nuclear matrix.
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PMID:Functional role of a highly repetitive DNA sequence in anchorage of the mouse genome. 341 21


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