Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.9 (enterokinase)
 675 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In a study of changes in digestive enzymes after massive intestinal resection and the mechanisms by which such changes occur, rats were sacrified 4 wk after removal of the proximal two-thirds of the small intestine. Alterations in the mucosal levels of sucrase, enterokinase, and dipeptide hydrolase (L-leucyl-L-alanine substrate) were examined in the light of associated changes in protein. DNA and wet mucosal weight, measured in standardized gut segments from various regions of intestine. Metabolic studies showed that normal growth patterns were reestablished after the operation but significant elevations in stool weight and fecal nitrogen occurred in the second postoperative week, falling towards normal by the 4th wk. In standard gut segments wet weight of mucosa, protein, and DNA rose, especially in distal segments, DNA increasing disproportionately. Mucosal levels of the proximally distributed and membrane-bound enzymes, sucrase and enterokinase, showed similar patterns of change: when enzyme activity was expressed in terms of the total per segment, proximally there were considerable increases in both enzymes, but, expressed in terms of specific activity, that of sucrase fell and that of enterokinase was unaltered. By contrast, the largely soluble and more distally distributed dipeptide hydrolase increased more in distal segments and the increases in total activity were accompanied by lesser increases in specific activity. However, in spite of increases in total activity, enzyme activity per milligram DNA fell by over 50% in postanastomotic segments. Subcellular distribution studies showed no change in the percentage of the total activity which was membrane-bound and zymograms confirmed that no new dipeptide hydrolase had appeared after resection. It is concluded that increases in the segmental totals of various enzymes seen after resection are achieved by disproportinate increases in the number of mucosal cells per segment and that the greatest change in a particular enzyme occurs in the region where the enzyme is normally found in highest concentration.
 ...
PMID:Changes in sucrase, enterokinase, and peptide hydrolase after intestinal resection. The association of cellular hyperplasia and adaptation. 469 57

The new aspect concerning the applicability of histidine and other affinity tags for the purification of oligomeric proteins, with particular emphasis on cleavage efficiency and final yield, is presented in this study. The final yield depends on both the cleavage efficiency and the degree of oligomerization of the protein. Cleavage procedures that are good enough for monomeric proteins can be problematic for oligomeric proteins. Random distribution of uncleaved or partially cleaved affinity tags among oligomers is the main cause of reduced yields. A trimeric protein, tumour necrosis factor alpha (TNF-alpha), bearing different histidine tags, was used as a model protein to explore and confirm this theoretical concept. Analysis of mixed TNF trimers, prepared from tag-free TNF doped with various amounts of histidine-tagged TNF, revealed an increased retention of the trimeric protein on immobilized metal-ion affinity chromatography (IMAC) columns. When 20% of histidine-tagged TNF was added, more than 50% of the protein was retained on the IMAC column. Thus, the applicability of histidine- and other affinity tags for purifying oligomeric proteins is significantly prejudiced in the case of higher oligomers. Various histidine-tags were fused to the N-terminus of full-length TNF-alpha and to the truncated form (dN6) of TNF-alpha. Two-step IMAC separation was used for purification. In the first step, IMAC-1, over 95% purity of histidine-tagged protein was achieved in all cases. Endo- and exoproteolytic removal of histidine tags with enterokinase (EKmax) and aminodipeptidase (DAPase) was studied and the major parameters affecting cleavage efficiency, microheterogeneity and final yield are critically discussed. IMAC-2 was used as the second and final step for removing the cleavage enzyme, cleaved tags, unprocessed protein and some other impurities. Selection of the optimal cleavage enzyme depends on the amino acid composition of the N-terminus and the intended use of the purified protein. The main conclusion is that special caution should be taken when introducing affinity tags to oligomeric proteins, with the final goal to produce pure, tag-free protein with acceptable yields. Given the same enzyme cleavage efficiency one can expect progressively reduced final protein yields with increasing degree of oligomerization. This should be considered as a general rule.
 ...
PMID:Influence of the protein oligomericity on final yield after affinity tag removal in purification of recombinant proteins. 1625 28

The new aspect concerning the applicability of histidine and other affinity tags for the purification of oligomeric proteins, with particular emphasis on cleavage efficiency and final yield, is presented in this study. The final yield depends on both the cleavage efficiency and the degree of oligomerization of the protein. Cleavage procedures that are good enough for monomeric proteins can be problematic for oligomeric proteins. Random distribution of uncleaved or partially cleaved affinity tags among oligomers is the main cause of reduced yields. A trimeric protein, tumour necrosis factor alpha (TNF-alpha), bearing different histidine tags, was used as a model protein to explore and confirm this theoretical concept. Analysis of mixed TNF trimers, prepared from tag-free TNF doped with various amounts of histidine-tagged TNF, revealed an increased retention of the trimeric protein on immobilized metal-ion affinity chromatography (IMAC) columns. When 20% of histidine-tagged TNF was added, more than 50% of the protein was retained on the IMAC column. Thus, the applicability of histidine- and other affinity tags for purifying oligomeric proteins is significantly prejudiced in the case of higher oligomers. Various histidine-tags were fused to the N-terminus of full-length TNF-alpha and to the truncated form (dN6) of TNF-alpha. Two-step IMAC separation was used for purification. In the first step, IMAC-1, over 95% purity of histidine-tagged protein was achieved in all cases. Endo- and exoproteolytic removal of histidine tags with enterokinase (EKmax) and aminodipeptidase (DAPase) was studied and the major parameters affecting cleavage efficiency, microheterogeneity and final yield are critically discussed. IMAC-2 was used as the second and final step for removing the cleavage enzyme, cleaved tags, unprocessed protein and some other impurities. Selection of the optimal cleavage enzyme depends on the amino acid composition of the N-terminus and the intended use of the purified protein. The main conclusion is that special caution should be taken when introducing affinity tags to oligomeric proteins, with the final goal to produce pure, tag-free protein with acceptable yields. Given the same enzyme cleavage efficiency one can expect progressively reduced final protein yields with increasing degree of oligomerization. This should be considered as a general rule.
 ...
PMID:Reprint of: Influence of the protein oligomericity on final yield after affinity tag removal in purification of recombinant proteins. 2189 2