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)

Prostaglandin H synthase (PGHS) catalyzes a key step in the biosynthesis of a variety of bioactive lipid mediators. The two known isoforms (PGHS-1 and -2) share about 60% amino acid identity, but exhibit distinct interactions with substrates, activators, and inhibitors. Ovine PGHS-1 has previously been shown to have a distinctive protease-sensitive site near Arg277; cleavage by trypsin, chymotrypsin, or proteinase K produces fragments of 33 and 38 kDa and loss of activity. The ovine PGHS-1 crystal structure shows Arg277 located in an exposed loop structure; homology modeling predicts similar loop structures for both human isoforms (hPGHS-1 and -2). We have used limited proteolytic digestion of recombinant hPGHS-1 and hPGHS-2 to probe their structures. Incubation of hPGHS-1 with either trypsin or proteinase K produced 33- and 38-kDa fragments and loss of activity. In contrast, incubation of hPGHS-2 with the same proteases led to cleavage of only a 2- to 3-kDa fragment, with no decrease in activity. Immunoblotting with site-specific antibodies demonstrated that the cleaved fragment originated from the hPGHS-2 C-terminus. Similar immunoblotting experiments indicated that trypsin did not attack the ovine PGHS-1 C-terminus. Mutagenesis was used to replace Pro263 of hPGHS-2 (corresponds to Arg277 of ovine PGHS-1) with arginine, inserting a potential trypsin site. Incubation of this P263R hPGHS-2 mutant with either trypsin or proteinase K resulted in cleavage near the C-terminus and retention of activity, just as with wild-type hPGHS-2. A peptide containing residues 259-268 of the P263R mutant was cleaved by trypsin at the same rate as a peptide corresponding to hPGHS-1 residues 272-281, demonstrating that the sequence differences were not responsible for the lack of tryptic cleavage at residue 263 in the hPGHS-2 mutant. Preincubation of hPGHS-2 with graded levels of guanidinium HCl before incubation with proteinase K did not produce large proteolytic fragments, indicating that the hPGHS-2 loop region was not selectively unfolding. The results point to two regions of significant structural difference between PGHS-1 and -2: the Arg277 loop, which is protease-sensitive in PGHS-1 but protease-resistant in PGHS-2, and the C-terminus, which is protease-sensitive in PGHS-2 but not in PGHS-1.
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PMID:Comparison of prostaglandin H synthase isoform structures using limited proteolytic digestion. 924 92

Rabbit monoclonal (RM) antibodies appear to have higher affinity for antigens than mouse monoclonal (MM) antibodies. However, RM antibodies have not been used in veterinary diagnostic immunohistochemistry. The authors compared reactivities of RM and MM antibodies on formalin-fixed, paraffin-embedded canine tissues, targeting 11 different antigens: CD3, CD79a, calcitonin, calretinin, chromogranin A, COX-2, estrogen receptor, Ki67, progesterone receptor, synaptophysin, and vimentin. Paraffin-embedded tissue sections were processed by 1 of 2 antigen-retrieval methods: 1) proteinase K digestion or 2) steam heat in citrate buffer. An additional set of slides did not receive antigen retrieval. Immunostaining was performed using an automated stainer, and scores were assigned to the different dilutions and antigen-retrieval methods on the basis of staining intensity and number of positive cells. Steam heat was usually the best antigen-retrieval method. The optimal dilution for each antibody was that which resulted in the highest specific staining and the lowest nonspecific (background) staining. The RM or MM antibodies yielded a specific reaction for all antigens examined except calretinin. The RM and MM antibodies yielded a specific reaction for 4 antigens only: COX-2, Ki67, synaptophysin, and vimentin. Three antigens (CD3, chromogranin A, and progesterone receptor) were detected only with RM antibodies, whereas the other 3 (CD79a, calcitonin, estrogen receptor) were detected only with MM antibodies. The results of this study differed from those reported for human tissues by the manufacturers of the antibodies. These results emphasize that, regardless of manufacturers' recommendations, each antibody must be individually standardized and validated before routine use in canine tissues.
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PMID:Comparison of rabbit monoclonal and mouse monoclonal antibodies in immunohistochemistry in canine tissues. 1613 Sep 92

Three COX-2-specific non-steroidal anti-inflammatory drugs (NSAIDs), etoricoxib, parecoxib, and nimesulide are widely prescribed against inflammatory conditions. However, their long term administration leads to severe conditions of cardiovascular complications and gastric ulceration. In order to minimize these side effects, C-terminal half (C-lobe) of colostrum protein lactoferrin has been indicated to be useful if co-administered with NSAIDs. Lactoferrin is an 80kDa glycoprotein with two similar halves designated as N- and C-lobes. Since NSAID-binding site is located in the C-terminal half of lactoferrin, C-lobe was prepared from lactoferrin by limited proteolysis using proteinase K. The incubation of lactoferrin with serine proteases for extended periods showed that N-lobe was completely digested but C-lobe was resistant for more than 72h indicating its long half life in the animal gut. The solution studies have shown that COX-2-specific NSAIDs bind to C-lobe with binding constants ranging from 10(-4) to 10(-5)M showing significant affinities for sequestering these compounds. In order to understand the mode of binding and sequestering properties, the complexes of C-lobe with all these three compounds, etoricoxib, parecoxib, and nimesulide were prepared and the structures of their complexes with C-lobe were determined at 2.2, 2.9, and 2.7A resolutions, respectively. The analysis of the structures of complexes of C-lobe with NSAIDs clearly show that all the three compounds bind firmly at the same ligand-binding site in the C-lobe revealing the details of the interactions between C-lobe and NSAIDs. The mode of binding of COX-2-specific NSAIDs to C-lobe is similar to that of the binding of COX-2 non-specific NSAIDs to C-lobe.
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PMID:Structural and binding studies of C-terminal half (C-lobe) of lactoferrin protein with COX-2-specific non-steroidal anti-inflammatory drugs (NSAIDs). 2051 46