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)

Complexes of HLA class II alpha- and beta-chains with invariant chain were proteolytically digested to study domain interactions between these molecules. Detergent extracts of metabolically labeled monensin-treated B lymphoblastoid cells (B-LCL) were digested with proteinase K and immunoprecipitated with anti-HLA-DR or anti-invariant chain antibodies. Subsequent two-dimensional polyacrylamide amide gel electrophoresis showed that proteinase K treatment results in the sequential generation of three polypeptides of approximately 21,500, 19,500, and 18,000 daltons respectively. All are proteolytic fragments derived from invariant chain, and all remain associated with class II antigens. Two-dimensional gels of endoglycosidase H-treated immunoprecipitates showed that all three fragments contain two N-linked oligosaccharides. Neuraminidase treatment of immunoprecipitates and Bandeiraea simplicifolia lectin binding of cell extracts showed that the largest fragment, but not the smallest fragment, also contains O-linked oligosaccharides. None of the fragments possess the transmembrane region; fragments were released in soluble form when biosynthetically labeled B-LCL were ruptured by freezing and thawing and intact membranes were separated from aqueous components by ultracentrifugation. Lack of the transmembrane sequence was confirmed on the 18,000 dalton fragment by demonstrating through specific peptide cleavage at tryptophanyl residues that this fragment retains a substantial portion of the C-terminal region of I chain beyond trp162. Retention of the C-terminal region excludes the presence of the transmembrane region when m.w. are considered. Our data, taken in context of the amino acid sequence of the invariant chain predicted by the cDNA clone, demonstrate that invariant chain interacts with class II antigens via its extracytoplasmic region.
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PMID:Invariant chain associates with HLA class II antigens via its extracytoplasmic region. 351 15

Buccal-cell-derived DNA collected by a 'swish and spit' technique and blood-derived DNA were compared for ease of collection, participant acceptance, utility and accuracy for HLA class II DR typing by the polymerase chain reaction with sequence-specific primers (PCR-SSP). The HLA class II DR typing results determined from DNA extracted by proteinase K digestion followed by phenol/chloroform extraction and ethanol precipitation from buccal cells and blood cells were identical for all subjects we studied (n = 10). Class II typing by PCR-SSP using DNA extracted from buccal cells stored at -20, 4, 25 or 37 degrees C for 1 week was successful. The samples can be collected without medical supervision and are not affected by exposure to a variety of temperature conditions for up to 1 week. The stability of the buccal cell specimens to these extreme conditions demonstrates the utility of this 'swish and spit' technique for collecting nucleated cells for geographically dispersed large-scale population studies. Buccal-cell-derived DNA collected by a simple 'swish and spit' mouthwash technique is an excellent and practical substitute for blood-derived DNA.
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PMID:A noninvasive 'swish and spit' method for collecting nucleated cells for HLA typing by PCR in population studies. 866 10

We used polymerase chain reaction (PCR)-based DNA typing to identify HLA class II alleles of two individuals from ancient human remains. Genomic DNAs were isolated from two ancient human skeletons excavated from the Sanganji and Kitakogane sites in the main and northern islands of Japan, respectively. They were archaeologically estimated to be approximately 5,000 and 6,000 years old respectively, representing the remnants from the Jomon era. High molecular weight DNA was extracted by the standard proteinase K-phenol extraction method followed by purification with a Centricon-30 micro concentrator. Several rounds of PCR successfully gave rise to amplification of the HLA-DRB1 and -DQA1 genes. The PCR-restriction fragment length polymorphism (PCR-RFLP) and sequencing based typing (PCR-SBT) methods revealed that those ancient individuals possessed the DRB1 and DQA1 alleles which are highly prevalent among the modern north Asian as well as Japanese populations.
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PMID:HLA genotyping of 5,000- and 6,000-year-old ancient bones in Japan. 1045 23