Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.9 (ribonuclease)
 6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using the method of isomer-specific proteolysis (ISP), the cis-trans nature of the peptide bonds involving prolines-114 and -117 in ribonuclease (RNase) has been investigated. These studies involve the pretreatment of RNase first with either a short pepsin pulse or a short mercaptoethanol pulse to irreversibly unfold the protein and then with a short chymotrypsin pulse to quickly cleave the Tyr115-Val116 bond so that the chain is suitably trimmed for the subsequent stereospecific cleavage either by aminopeptidase P, to investigate proline-117, or by a proline-specific endopeptidase, to investigate proline-114. The most reasonable interpretation of our results suggests that proline-117 is essentially 100% trans in both the native and unfolded states, so it apparently makes no direct contribution to the slow refolding kinetics of RNase. It is also determined that proline-114 is 100% cis in native RNase and ca. 95% cis in reversibly unfolded RNase so only 5% of the unfolded RNase can be rate limited by trans to cis isomerization of proline-114 during refolding. Careful spectroscopic studies of refolding show that the smallest and slowest of the refolding phases, the ct phase, has the proper amplitude (5%), relaxation time (400 s at 10 degrees C), and activation energy (17 kcal) for a phase that is rate limited by the trans to cis isomerization of proline-114. Measurements of the kinetics of binding of cytidine 2'-monophosphate during refolding further show that RNase does not become active until proline-114 has isomerized to the native cis configuration. It is concluded that none of the three prolines thus far examined (i.e., prolines-93, -114, and -117) by the ISP method is involved in the formation of a fully active, nativelike intermediate which has "incorrect" proline isomers. The specific structural process which is responsible for the largest of the three slow refolding phases, the XY phase, is still undetermined. Although ISP results on proline-42 are not yet available, it seems possible that this slow phase may be rate limited by a process other than proline isomerization. In unrelated studies, results from chymotrypsin hydrolyses of several short peptides containing the sequence -X-Y-Pro- show that cleavage of an active X-Y bond is very slow when it is immediately adjacent on the amino side of a proline peptide bond. Thus, chymotrypsin cleavage may not be generally useful as the analytical step in isomer-specific proteolysis.
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PMID:Involvement of prolines-114 and -117 in the slow refolding phase of ribonuclease A as determined by isomer-specific proteolysis. 644 92

Several studies in the past few years have supported the hypothesis that oxytocin (OT) is synthesized in a paracrine system within the pregnant human uterus and that this paracrine system may be an important regulator of the timing of human parturition. Using ribonuclease protection assays, we have demonstrated a three-fold increase in the rate of synthesis of OT mRNA in human decidua around the time of parturition. We also have shown that a similar increase in OT mRNA and peptide synthesis can be stimulated in vitro by physiological concentrations of estradiol. This increase is inhibited by concomitant use of the estrogen receptor (ER) blocker tamoxifen or by transcription inhibitors. Progesterone had little, if any effect. We also detected mRNAs for ER and progesterone receptor (PR) in amnion, chorion and decidua with the same relative tissue concentrations as OT mRNA. The concentrations of ER but not PR increased significantly around the time of labour onset. To determine if local OT concentrations may be regulated by changes in OT metabolism, we determined kinetic parameters for OT metabolism in decidua, chorion and placenta. [3H]tyrosyl-OT was used as substrate. Metabolites were separated using HPLC and identified using amino acid analysis and mass spectrometry. Metabolism in decidua and chorion occurred predominantly via a cytosolic post-proline endopeptidase and the activity was comparable to placenta. In microsomal fractions, cystine aminopeptidase activity predominated and placenta had significantly more activity than decidua and chorion. There were no changes in any Km or apparent vmax values around the time of parturition. These findings support the existence of a paracrine system within human decidua that involves sex steroids regulating synthesis of OT and that undergoes significant changes around the time of parturition. Changes in local OT concentrations are controlled by rates of synthesis rather than rates of metabolism.
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PMID:Synthesis and metabolism of oxytocin in late gestation in human decidua. 871 92