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Literature summary for 1.3.1.70 extracted from
- Microsomal enzymes of cholesterol biosynthesis from lanosterol. Characterization, solubilization, and partial purification of NADPH-dependent DELTA8,14-steroid 14-reductase (1984), J. Biol. Chem., 259, 13413-13423.
Activating Compound
| Activating Compound | Comment | Organism | Structure |
|---|---|---|---|
| phosphatidylcholine | liposomal phosphatidylcholine, egg yolk: 1.4fold activation | Rattus norvegicus |  |
| Phospholipids | partially purified enzyme: activation | Rattus norvegicus | |
Application
| Application | Comment | Organism |
|---|---|---|
| analysis | partial purified enzyme suitable for reconstitution studies, reconstitution of isolated, soluble enzymes involved in cholesterol biosynthesis from lanosterol | Rattus norvegicus |
General Stability
| General Stability | Organism |
|---|---|
| detergents such as Lubrol-WX, Emulgen 911, Triton X-100, Triton WR-1339, and their combinations with ionic detergents such as taurodeoxycholate, cholate, and Na+ salts destroy enzyme even at low concentration, 0.1-0.5%, w/v | Rattus norvegicus |
| enzyme in soluble form is very instable | Rattus norvegicus |
| stable against trypsin treatment | Rattus norvegicus |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| Ca2+ | 4 mM: very slight, 3.5% inhibition | Rattus norvegicus | |
| cyanide | cyanide ion, membrane-bound enzyme, modest inhibition at high concentrations, greater than 1 mM, partial purified enzyme, sodium cyanide, no or very poor inhibition, 10 mM: 3.5% inhibition | Rattus norvegicus | |
| HgCl2 | 3 mM: more than 99% inhibition, inhibition negligible in the presence of 10 mM glutathione | Rattus norvegicus | |
| Mg2+ | 4 mM: slight, 6% inhibition | Rattus norvegicus | |
| additional information | no direct inhibition by 3beta-hydroxy-4,4-dimethyl-5alpha-cholest-8(14)-en-15-one, even when concentration is increased to level of Km for substrate; no inhibition by trypsin | Rattus norvegicus | |
| N-ethylmaleimide | 5 mM: 53% inhibition, inhibition negligible in the presence of 10 mM glutathione | Rattus norvegicus | |
| O2 | 28% inhibition | Rattus norvegicus | |
| Phospholipase A2 | very high inhibition by very small concentrations, activity not restored by addition of phospholipids or any components presumed to be released from treated microsomes. Various concentration ranges of potential cofactors including FMN, FAD, AMP, ADP, ATP, coenzyme Q, Coenzyme A, and hemin, plus heat-treated microsomes, and lipid extracts of microsomes does not restore microsomal 14-reductase of phospholipase A2-treated microsomes | Rattus norvegicus | |
| sulfhydryl-binding agents | - |
Rattus norvegicus | |
| trans-1,4-bis(2-chlorobenzylaminomethyl)cyclohexane dihydrochloride | AY-9944, strong inhibition, 0.0003 mM: 50% inhibition, 0.0005 mM: 65% inhibition | Rattus norvegicus | |
| Triparanol | 4-chloro-alpha-[4-[2-diethylaminoethoxy]phenyl]-alpha-(4-methylphenyl)benze-methanol, slight inhibition | Rattus norvegicus | |
| Tris-HCl | Tris-HCl buffer, 28% inhibition, not restored by addition of KCl at various concentrations | Rattus norvegicus | |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | kinetics | Rattus norvegicus | |
| additional information | - |
additional information | kinetic data | Rattus norvegicus | |
| 0.0294 | - |
4,4-dimethyl-5alpha-cholesta-7,14-dien-3beta-ol | membrane-bound enzyme, Lineweaver-Burk | Rattus norvegicus | |
| 0.0333 | - |
4,4-dimethyl-5alpha-cholesta-7,14-dien-3beta-ol | soluble enzyme | Rattus norvegicus | |
| 0.0345 | - |
4,4-dimethyl-5alpha-cholesta-7,14-dien-3beta-ol | microsomal-bound enzyme | Rattus norvegicus | |
| 0.6 | - |
NADPH | membrane-bound enzyme | Rattus norvegicus | |
Localization
| Localization | Comment | Organism | GeneOntology No. | Textmining |
|---|---|---|---|---|
| endoplasmic reticulum | - |
Rattus norvegicus | 5783 | - |
| membrane | membrane-bound | Rattus norvegicus | 16020 | - |
| microsome | microsomal-bound | Rattus norvegicus | - |
- |
| additional information | distribution | Rattus norvegicus | - |
- |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| additional information | no activation by Mg2+ or Ca2+, 0.5-20 mM, but slight inhibition | Rattus norvegicus |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 4,4-dimethyl-5alpha-cholesta-8,14-dien-3beta-ol + NADPH | Rattus norvegicus | presumably the natural substrate | 4,4-dimethyl-5alpha-cholesta-8-en-3beta-ol + NADP+ | - |
? | |
| additional information | Rattus norvegicus | catalyzes anaerobically NADPH-dependent reduction of 14-double bond of 8,14-diene or 7,14-diene sterols that are sterol intermediates formed during C-32 demethylation in cholesterol biosynthesis from lanosterol in mammals | ? | - |
? | |
| additional information | Rattus norvegicus | reduction of 14-double bond of sterol-conjugated dienes | ? | - |
? | |
| additional information | Rattus norvegicus | microsomal enzyme of cholesterol biosynthesis from lanosterol | ? | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Rattus norvegicus | - |
male sprague-dawley rats | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| solubilization with combined octylglucoside and sodium taurodeoxycholate system and partial purification | Rattus norvegicus |
Reaction
| Reaction | Comment | Organism | Reaction ID |
|---|---|---|---|
| 4,4-dimethyl-5alpha-cholesta-8,24-dien-3beta-ol + NADP+ = 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol + NADPH + H+ | reduction of 14-double bond of conjugated DELTA8,14-sterols and DELTA7,14-sterols | Rattus norvegicus |
Source Tissue
| Source Tissue | Comment | Organism | Textmining |
|---|---|---|---|
| liver | - |
Rattus norvegicus | - |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 0.0555 | - |
partially purified enzyme | Rattus norvegicus |
Storage Stability
| Storage Stability | Organism |
|---|---|
| -80°C, 1 week, stable | Rattus norvegicus |
| 4°C, overnight, 30% activity lost | Rattus norvegicus |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 4,4-dimethyl-5alpha-cholesta-7,14-dien-3beta-ol + NADPH | 64% activity compared with 4,4-dimethyl-5alpha-cholesta-8,14-dien-3beta-ol | Rattus norvegicus | 4,4-dimethyl-5alpha-cholesta-7-en-3beta-ol + NADP+ | - |
? | |
| 4,4-dimethyl-5alpha-cholesta-8,14-dien-3beta-ol + NADPH | best substrate, probably natural substrate | Rattus norvegicus | 4,4-dimethyl-5alpha-cholesta-8-en-3beta-ol + NADP+ | - |
? | |
| 4,4-dimethyl-5alpha-cholesta-8,14-dien-3beta-ol + NADPH | presumably the natural substrate | Rattus norvegicus | 4,4-dimethyl-5alpha-cholesta-8-en-3beta-ol + NADP+ | - |
? | |
| 5alpha-cholesta-7,14-dien-3beta-ol + NADPH | 43% activity compared with 4,4-dimethyl-5alpha-cholesta-8,14-dien-3beta-ol | Rattus norvegicus | 5alpha-cholesta-7-en-3beta-ol + NADP+ | - |
? | |
| 5alpha-cholesta-8,14-dien-3beta-ol + NADPH | DELTA8,14-cholestadien-3beta-ol | Rattus norvegicus | 5alpha-cholesta-8-en-3beta-ol + NADP+ | - |
? | |
| 5alpha-cholesta-8,14-dien-3beta-ol + NADPH | 53% activity compared with 4,4-dimethyl-5alpha-cholesta-8,14-dien-3beta-ol | Rattus norvegicus | 5alpha-cholesta-8-en-3beta-ol + NADP+ | - |
? | |
| additional information | substrate specificity | Rattus norvegicus | ? | - |
? | |
| additional information | enzyme acts on a range of steroids with a 14(15)-double bond | Rattus norvegicus | ? | - |
? | |
| additional information | reduction of 14-double bond of conjugated DELTA8,14-diene sterols and DELTA7,14-diene sterols | Rattus norvegicus | ? | - |
? | |
| additional information | sterol substrate specificity study | Rattus norvegicus | ? | - |
? | |
| additional information | anaerobic conditions yield maximal rates of reduction | Rattus norvegicus | ? | - |
? | |
| additional information | 4-nor-methyldiene sterols, 5alpha-cholesta-8,14-dien-3beta-ol, and 5alpha-cholesta-7,14-dien-3beta-ol are reduced at about 50% of the rate of the corresponding 4-gem-dimethyldiene sterols, for both the C27-sterol and C29-sterol, the higher reaction rate of the 8,14-diene system is consistently observed over the isomeric 7,14-diene | Rattus norvegicus | ? | - |
? | |
| additional information | catalyzes anaerobically NADPH-dependent reduction of 14-double bond of 8,14-diene or 7,14-diene sterols that are sterol intermediates formed during C-32 demethylation in cholesterol biosynthesis from lanosterol in mammals | Rattus norvegicus | ? | - |
? | |
| additional information | reduction of 14-double bond of sterol-conjugated dienes | Rattus norvegicus | ? | - |
? | |
| additional information | microsomal enzyme of cholesterol biosynthesis from lanosterol | Rattus norvegicus | ? | - |
? | |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 37 | - |
assay at, anaerobically | Rattus norvegicus |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7.4 | - |
pH-optimum and assay at, anaerobically | Rattus norvegicus |
pH Range
| pH Minimum | pH Maximum | Comment | Organism |
|---|---|---|---|
| additional information | - |
14-reductase has relatively broad pH profile in acidic region, activity drastically decreases with only moderate elevation of pH above 7.8 | Rattus norvegicus |
| 7.8 | - |
14-reductase has relatively broad pH profile in acidic region, activity drastically decreases with only moderate elevation of pH above 7.8 | Rattus norvegicus |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| additional information | NADH only poorly supports reduction | Rattus norvegicus | |
| NADPH | NADPH-dependent | Rattus norvegicus | |
| NADPH | NADPH required, NADH has 4.5%, of the NADPH activity | Rattus norvegicus | |
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