Comparison of the 17α-Hydroxylase/C17,20-Lyase Activities of Porcine, Guinea Pig and Bovine P450c17 Using Purified Recombinant Fusion Proteins Containing P450c17 Linked to NADPH-P450 Reductase
Bibliographic record
Abstract
The cDNAs for cytochrome P450c17 (P450c17) of three species, pig, guinea pig, and cow, representing three families of mammals (suidae, procaviidae, and bovidae, respectively) were each engineered into an expression plasmid (pCWori+). The P450c17 domain of the coding sequence was connected to a truncated form of rat NADPH-P450 reductase by a linker sequence encoding two amino acids (SerThr). These fusion proteins were expressed in E. coli and purified for use in enzymatic assays to determine similarities and differences in 17 alpha-hydroxylase and lyase activities. The fusion proteins were found to catalyze both the 17 alpha-hydroxylation of progesterone (P4) and pregnenolone (P5) to 17 alpha-hydroxylated P4 and P5 (17 alpha-OH P4 and 17 alpha-OH P5) followed by the C17,20-lyase reaction for the conversion of these C(21)-17 alpha-hydroxylated steroids to C(19)-steroids (the C17,20-lyase reaction). These in vitro studies show that (a) porcine P450c17 possesses cytochrome b(5) (b(5))-stimulated C17,20-lyase activity that converts 17 alpha OH-P4 to androstenedione (AD) but also converts 17 alpha-OHP5 to dehydroepiandrosterone (DHEA); (b) guinea pig P450c17 possesses a b(5)-stimulated C17,20-lyase activity that converts 17 alpha-OH P4 to AD but does not convert 17 alpha-OH P5 to DHEA., and (c) bovine P450c17 possesses a b(5)-stimulated C17,20-lyase activity that converts 17 alpha-OH P5 to DHEA but does not convert 17 alpha-OH P4 to AD. Thus, the P450c17 of each species differs in its ability to catalyze in vitro the conversion of C(21)-steroids to C(19)-steroids. In addition, each P450c17 is capable of catalyzing additional hydroxylation reactions leading to low levels of 2 alpha-, 6 beta-, 16- and 21-hydroxy-metabolites. Porcine P450c17 also catalyzes the b(5)-dependent synthesis of andien-beta (androsta-5,16-dien-3beta-ol) from P5. When the amino acid sequences of the three P450c17s were aligned there was an approximate 50% variation in the alignment identity (227 differences in the sequences of 509 amino acids). Alignment did not permit the assignment of specific amino acids or domains to the observed differences in enzymatic activities.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.001 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".