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Made available in DSpace on 2014-06-11T19:35:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-02-15Bitstream added on 2014-06-13T20:06:31Z : No. of bitstreams: 1 felippe_lg_dr_araiq.pdf: 5698619 bytes, checksum: b7407447386c76e0ea7905f1c8a0f7cf (MD5) / O estudo fitoquímico precedente da espécie Peperomia blanda culminou no isolamento de lignanas tetraidrofurânicas e secolignanas. Alguns isômeros destas secolignanas apresentaram configurações absolutas diferentes daquelas já relatadas, as quais foram determinadas por comparação dos espectros experimentais de dicroísmo circular vibracional e eletrônico com aqueles preditos por cálculos mecânico-quânticos ab initio. A peperomina B teve a configuração absoluta assinalada como (2S,3S,5S), confirmando a estrutura química descrita na literatura, a substância 2-metil-3-[bis(3′,4′-metilenodioxi-5′-metoxifenil) metil]butirolactona foi assinalada como (2R,3S) e a substância 2-metil-3-[5-(3′,4′-metilenodioxi-5′-metoxifenil)-5-(3′,4′,5′-trimetoxifenil)metil]butirolactona foi assinalada como (2R,3S,5S). As dinâmicas metabólicas sazonais de três lignanas tetraidrofurânicas e três secolignanas foram determinadas a partir de análise por CLAE-DAD dos extratos metanólicos de partes aéreas e raízes de duas populações de P. blanda, utilizando planejamento experimental e validação do método cromatográfico. O maior acúmulo destas substâncias foi observado nas raízes, especialmente nos meses de julho e em espécies coletada na Reserva Florestal da Ripasa. Alguns fatores climáticos, como a disponibilidade hídrica, foram determinantes para a produção destes metabólitos. Uma das lignanas tetraidrofurânicas, com oxidação na posição C-9, bem como as secolignanas, tiveram alguns aspectos biossintéticos abordados pela incorporação de precursores em extratos enzimáticos. Os estudos das vias biossintéticas envolvidas na formação dessas lignanas foram iniciados com a investigação e otimização das reações catalíticas da enzima fenilalanina amônia liase (PAL). A condição ótima reacional foi determinada por... / The previous phytochemical study of Peperomia blanda species culminated in the isolation of secolignans and tetrahydrofuran lignans. Some of these secolignans isomers showed different absolute configurations from those reported in the literature, which were determined by comparison of experimental vibrational and electronic circular dichroism spectra with those calculated using ab initio quantum mechanical methods. The stereochemistry of peperomin B was assigned as (2S,3S,5S) confirming the previous related structure, the compound 2-methyl-3-[bis(3′,4′-methylenedioxy-5′-methoxyphenyl)methyl] butyrolactone was assigned as (2R,3S) and the compound 2-methyl-3-[5-(3′,4′-methylenedioxy-5′-methoxyphenyl)-5-(3′,4′,5′-trimethoxyphenyl)methyl] butyrolactone was assigned as (2R,3S,5S). The seasonal metabolic dynamic of three tetrahydrofuran lignans and three secolignans were determined by HPLC-DAD analysis of methanolic extracts of aerial parts and roots of two populations of P. blanda, using experimental design and validation of the chromatographic method. The largest accumulation of these substances was observed in the roots, specially in July and in the species collected in the Ripasa's Forest Reserve. Some climatic factors such as water availability were critical for the production of these metabolites. One of the tetrahydrofuran secolignans with oxidation at C-9 as well as the secolignans were studied from the biosynthetic point of view using cell free extracts. These studies were started with the investigation and optimization of catalytic reactions of the enzyme phenylalanine ammonia lyase (PAL). The optimum reaction condition was determined by experimental design which showed better conversion of L-phenylalanine to trans-cinnamic acid at 51 °C and pH 9.2. The... (Complete abstract click electronic access below)

Made available in DSpace on 2016-08-12T18:48:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-10-02. Added 1 bitstream(s) on 2016-08-12T18:51:07Z : No. of bitstreams: 1 000857400_20161029.pdf: 742571 bytes, checksum: 487ad640c375e955b44e53a46dcc2619 (MD5) Bitstreams deleted on 2016-10-31T10:32:36Z: 000857400_20161029.pdf,. Added 1 bitstream(s) on 2016-10-31T10:33:10Z : No. of bitstreams: 1 000857400.pdf: 3598805 bytes, checksum: edaed29b3d2a17a9e87b4d7c4ad65759 (MD5) / Piper arboreum é conhecida por acumular amidas pirrolidínicas com potente atividade antifúngica, antileishimanicida, inseticida entre outros, sendo, portanto, importantes substâncias alvos para estudos biossintéticos. À partir dos extratos das folhas e frutos desta espécie, foram isoladas três amidas pirrolidínicas e dois precursores fenilpropanoídicos, sendo que piperilina foi utilizada como padrão para os estudos biossintéticos. A proposta biossintética para a formação dessas amidas segue uma biossíntese mista, que deriva da via do chiquimato e via do acetato. Unidades C6-C3, condensam-se com malonil-CoA e a ornitina para a produção destes metabólitos. Os estudos biossintéticos foram iniciados com a incorporação de CH313CO2Na em folhas de P. arboreum. Este experimento teve como objetivo estabelecer a biossíntese da extensão final da cadeia lateral das amidas pirrolidínicas em relação ao derivado fenilpropanoídico. A análise por espectrometria de massas confirmou a incorporação do isótopo 13C na piperilina, indicando que a extensão da cadeia ocorre via acetato. Para verificar se a formação do grupo metilenodioxílico ocorre antes ou depois da formação da porção amídica, foram sintetizados os precursores fenilpropanoídicos: o ácido ferúlico e o 3',4'-metilenodioxicinâmico, bem como seus análogos marcados com isótopos estáveis: o ácido[1-OD, 2-D]-ferúlico e o ácido[1-OD, 2-D]- 3',4'- metilenodioxicinâmico. Os precursores sintetizados marcados com deutério foram incorporados, separadamente, na fração enzimática solúvel, juntamente com os precursores L-ornitina, e ácido malônico. Os produtos formados foram monitorados por CLAE-DAD utilizando-se a piperilina como padrão. A formação do produto foi identificado por CLAE-DAD. A análise do extrato protéico das folhas e frutos em gel poliacrilamida permitiu atribuir as bandas mais expressivas as... / Piper arboreum is known to accumulate amides with potential antitumoral, antimicrobial, antifungal, antileishmanial and insecticidal activities, being targeted compounds for biosynthetic studies. From leaves and fruits extracts, three pyrrolidinic amides and two phenylpropanoids precursors were isolated and the piperilina amide was used as standard for biosynthetic studies. The biosynthetic proposal for piperiline precusors formation follows a mixed biosynthesis, which is derived from the shikimate and acetato are which C6-C3 units which condense with malonyl-CoA and ornithine for the production of the amides. The biosyntetic studies were initiated with the incorporation of CH313CO2Na in leaves of P. arboreum. Analysis by mass spectrometry confirmed incorporation of 13C isotope indicating that extention chain of piperilina is product via acetate. To determine formation of the methylenedioxy group occurs before or after the amide portion, were if the synthesized phenylpropanoids precursors ferulic acid and 3',4'-methylenedioxycynamic acid as well the same compounds labeled with stable isotopes: [1-OD, 2-D]-ferulic acid and [1-OD, 2-D]- 3',4'-methylenedioxycynamic acid. The synthetized labeled precursors were incorpored into the soluble fraction of the enzymatic extraction, together with precursors L-ornithine and malonic acid. The products were analyzed by liquid chromatography and mass espectrometry using piperline amide as standard. Analysis of the protein extract from de leaves and fruits using 1D polyacrylamide gel allowed to assing the most significant bands: the RuBisCo (55 - 14 kDa), PAL (77- 83 kDa) and PKS (45 - 30 kDa) enzimes. The piperiline amide isolated from P. arboreum were submetted to inativation assay of the enzyme tyrosinase and showed powerful in vitro activity. The etanolic extracts from leaves from P. arboreum showed important antifungal, anti-inflammatory and inseticidal...

Orientador: Maysa Furlan / Banca: Nivaldo Boralle / Banca: Hosana Maria Debonsi / Banca: Marcos Pivatto / Banca: Alaide de Sá Barreto / Resumo: Piper arboreum é conhecida por acumular amidas pirrolidínicas com potente atividade antifúngica, antileishimanicida, inseticida entre outros, sendo, portanto, importantes substâncias alvos para estudos biossintéticos. À partir dos extratos das folhas e frutos desta espécie, foram isoladas três amidas pirrolidínicas e dois precursores fenilpropanoídicos, sendo que piperilina foi utilizada como padrão para os estudos biossintéticos. A proposta biossintética para a formação dessas amidas segue uma biossíntese mista, que deriva da via do chiquimato e via do acetato. Unidades C6-C3, condensam-se com malonil-CoA e a ornitina para a produção destes metabólitos. Os estudos biossintéticos foram iniciados com a incorporação de CH313CO2Na em folhas de P. arboreum. Este experimento teve como objetivo estabelecer a biossíntese da extensão final da cadeia lateral das amidas pirrolidínicas em relação ao derivado fenilpropanoídico. A análise por espectrometria de massas confirmou a incorporação do isótopo 13C na piperilina, indicando que a extensão da cadeia ocorre via acetato. Para verificar se a formação do grupo metilenodioxílico ocorre antes ou depois da formação da porção amídica, foram sintetizados os precursores fenilpropanoídicos: o ácido ferúlico e o 3',4'-metilenodioxicinâmico, bem como seus análogos marcados com isótopos estáveis: o ácido[1-OD, 2-D]-ferúlico e o ácido[1-OD, 2-D]- 3',4'- metilenodioxicinâmico. Os precursores sintetizados marcados com deutério foram incorporados, separadamente, na fração enzimática solúvel, juntamente com os precursores L-ornitina, e ácido malônico. Os produtos formados foram monitorados por CLAE-DAD utilizando-se a piperilina como padrão. A formação do produto foi identificado por CLAE-DAD. A análise do extrato protéico das folhas e frutos em gel poliacrilamida permitiu atribuir as bandas mais expressivas as... / Abstract: Piper arboreum is known to accumulate amides with potential antitumoral, antimicrobial, antifungal, antileishmanial and insecticidal activities, being targeted compounds for biosynthetic studies. From leaves and fruits extracts, three pyrrolidinic amides and two phenylpropanoids precursors were isolated and the piperilina amide was used as standard for biosynthetic studies. The biosynthetic proposal for piperiline precusors formation follows a mixed biosynthesis, which is derived from the shikimate and acetato are which C6-C3 units which condense with malonyl-CoA and ornithine for the production of the amides. The biosyntetic studies were initiated with the incorporation of CH313CO2Na in leaves of P. arboreum. Analysis by mass spectrometry confirmed incorporation of 13C isotope indicating that extention chain of piperilina is product via acetate. To determine formation of the methylenedioxy group occurs before or after the amide portion, were if the synthesized phenylpropanoids precursors ferulic acid and 3',4'-methylenedioxycynamic acid as well the same compounds labeled with stable isotopes: [1-OD, 2-D]-ferulic acid and [1-OD, 2-D]- 3',4'-methylenedioxycynamic acid. The synthetized labeled precursors were incorpored into the soluble fraction of the enzymatic extraction, together with precursors L-ornithine and malonic acid. The products were analyzed by liquid chromatography and mass espectrometry using piperline amide as standard. Analysis of the protein extract from de leaves and fruits using 1D polyacrylamide gel allowed to assing the most significant bands: the RuBisCo (55 - 14 kDa), PAL (77- 83 kDa) and PKS (45 - 30 kDa) enzimes. The piperiline amide isolated from P. arboreum were submetted to inativation assay of the enzyme tyrosinase and showed powerful in vitro activity. The etanolic extracts from leaves from P. arboreum showed important antifungal, anti-inflammatory and inseticidal... / Doutor

UDP-glucose, the precursor for cellulose biosynthesis, can be produced via the catalysis of sucrose by sucrose synthase (SuSy) or through the phosphorylation of glucose-I-phosphate by UDP-glucose pyrophosphorylase (UGPase). As such, these genes, together with sucrose phosphate synthase (SPS) which recycles fructose (an inhibitor of SuSy), are interesting targets for altering carbon allocation in plants. In an attempt to alter cell wall biosynthesis in plants, targeted overexpression of SuSy, UGPase and SPS independently and in a pyramiding strategy was assessed in tobacco. All lines displayed enhanced growth and biomass production, and in the case of double and triple transgenics, there was an additive effect. Despite the increased growth rates, there was no consistent change in soluble carbohydrate pools. Furthermore, only the triple transgenics had constant changes in structural carbohydrates: with increased hemicellulose content and slight increases in cellulose. Collectively, these results support the role of SPS, SuSy and UGPase in maintaining sink strength, but suggest that the reallocation of carbon to cellulose production in tobacco may not be possible by overexpressing these genes. In contrast, transgenic poplar overexpressing UGPase produced significantly more cellulose than wild-type trees. However, this was accompanied by a severe reduction in growth and the production of a salicylic acid glucoside (SAG) in significant quantities. The UDP-glucose generated by UGPase overexpression appeared to participate in both the synthesis of cellulose and SAG, suggesting that cellulose biosynthesis may be limited by the cellulose synthase complex. Poplar transformed with SuSy and with SuSy x UGPase also had increased cellulose production. The trees were phenotypically normal, with only minor reductions in height growth in some lines. It appears that UDP-glucose may be channelled directly to the cellulose synthase complex by SuSy. The increased cellulose content was associated with an increase in cell wall crystallinity, but there was no change in microfibril angle, confirming the re-allocation to cellulose synthesis was not the result of tension wood formation, again supporting the hypothesis that the cellulose synthase complex is the limiting factor. Clearly, it is possible to alter cellulose deposition in trees by augmenting sucrose metabolism to produce UDP-glucose, the precursor to cellulose biosynthesis. / Forestry, Faculty of / Graduate

The biosynthetic origin of the plant growth regulator abscisic acid remains equivocal and almost nothing is known about the enzymes involved in this process. The present research programme describes the development of a cell-free system, capable of synthesizing abscisic acid and attempts to provide further information about the biochemistry and enzymology of this important biosynthetic pathway. Cell-free extracts were prepared either directly from the flavedo (crude) or from an acetone powder derived from flavedo, of mature coloured fruits of Citrus sinensis L. cv. Midknight and incubated with mevalonic acid, isopentenyl pyrophosphate, famesylpyrophosphate, geranylgeranyl pyrophosphate, ß-carotene and 1',4'-trans-abscisic acid diol. The neutral and acidic products formed were purified by thin-layer chromatography and high performance liquid chromatography, and quantified by high performance liquid chromatography, gas chromatography-electron capture and unequivocally identified by combined gas chromatography-mass spectrometry. Abscisic acid, 1',4'-trans-abscisic acid diol and phaseic acid were unequivocally identified as the major acidic products formed in this cell-free system. The acid fraction also contained xanthoxin acid. Labelled and unlabelled ß-carotene was converted into the neutral compounds xanthoxin and xanthoxin alcohol. In addition. high performance liquid chromatography-photodiode array analYSis of the oxy-carotenoid fraction revealed the complete spectrum of ß, ß-carotenoids induding zeaxanthin, antheraxanthin and violaxanthin with accumulation of an oxygenated carotenoid tentatively identified as 9- cis-violaxanthin. Identification of putative C₁₅ intermediates was achieved by either UV spectrophotometry and combined capillary gas chromatography-mass spectrometry or microchemical analYSis and co-chromatography. Refeeding studies using (±)-[2-¹⁴C]_ abscisic acid diol as substrate revealed that abscisic acid was not metabolized to abscisic acid diol, suggesting that it was/is produced as an intermediate rather than as a catabolite of ABA in this system. Stigmasterol, and to a lesser extent cholesterol reduced conversion of ß-carotene to abscisic acid but did not influence transformation of 1',4'-trans-abscisic acid diol to abscisic acid. AM01618 stimulated fonnation of abscisic acid and appeared to exert its effect at the level of conversion of 1' ,4'-trans-abscisic acid diol. Zeatin and the cytokinin analogue, ancymidol inhibited the biosynthesis of abscisic acid whereas dithiothreitol increased incorporation of label from ß-carotene into abscisic acid suggesting involvement of a cytochrome P450-type mixed function oxidase in this reaction sequence. Sodium dodecylsulphate polyacrylamide gel electrophoresis of the enzyme extract derived from Citrus flavedo revealed the presence of a 53 kD protein with peroxidase activity characteristic of a cytochrome P-450. Abscisic acid biosynthesizing activity was always greater in extracts from acetone powder and abscisic acid biosynthesis was enhanced in the presence of AMO 1618, NAD+, NADH, NADPH, MgCI₂ and Molybdate but was inhibited by FAD. Activity was further enhanced by the addition of (R,S)-abscisic acid as a cold-pool trap and by induding 0.1% w/v of either Tween 20 or Triton X 100 in the extraction buffer. When cis-ß-carotene was used as substrate, no abscisic acid was produced. Conversely when either all-trans-ß-carotene or a mixture of the two isomers was used, incorporation into abscisic acid occurred. Upoxygenase activity in cell-free extracts of Citrus flavedo increased with increasing protein concentration. As the ability of lipoxygenase to make xanthoxin from violaxanthin, had been reported, increased activity in the cell-free system implied that carotenoid deavage was being brought about by a non-haem oxygenase with lipoxygenase-like properties. Reports had implicated phoshorylation in the activation of many catalytic enzymes (Hanks et aI., 1985). Phosphorylation of the enzymes in this cell-free system proved unsuccessful. Further, it had been reported that in vitro phosphorylation of several membrane polypeptides and soluble polypeptides from com, had been promoted by the addition of Ca²₊ In this cell-free system Ca + did not have a stimulatory effect on protein phosphorylation. Dioxygenases generally occur as soluble enzymes, where they catalyse many oxygenation reactions in metabolic pathways. The addition of 2-oxo-glutarate, a requirement of most soluble oxidases, did not affect the activity of the cell-free system.

M.Sc. / On exposure to abiotic or biotic stresses, plants initiate a cascade of metabolic reactions, some of which lead to the biosynthesis of secondary metabolites with roles in self defense. Phytoalexins are a class of secondary metabolites synthesized de novo in response to microbial attack by activation of certain biosynthetic pathways. Cruciferae phytoalexins are all indole based with a carbon, nitrogen and sulfur containing constituent on the 3’ position of the indole ring. This common similarity of all Cruciferae phytoalexins suggests that the plants all share a common indole precursor. Camalexin is the primary phytoalexin of Arabidopsis thaliana. De novo synthesis of camalexin upon infection, as well as its antimicrobial nature supports its role in disease resistance. Evidence exists that suggests the inducible biosynthesis of camalexin involves steps of the tryptophan pathway, along with an increase in transcript and protein levels of the tryptophan pathway enzymes after microbial infection. Bacterial LPS (lipopolysaccharide) has been described as one of the microbe/pathogenassociated molecular patterns (M/PAMPs) capable of eliciting the activation of the plant innate immune system. LPS is an integral component of the cell surface of Gram-negative bacteria. It is a complex which is exposed to the external environment, and is thus involved with external interactions of the bacteria. The hypothesis investigated in this dissertation is that LPS, as a lipoglycan PAMP, results in activation of signal transduction pathways involved in defense that lead to the production of the defense metabolite, camalexin. Furthermore, that the genes CYP71B15, CYP79B2 and TSB are up-regulated in response to LPS during camalexin biosynthesis via the tryptophan pathway. To test this hypothesis, camalexin production was investigated through a combination of analytical techniques including thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography (GC), ultra pressure liquid chromatography-mass spectrometry (UPLC-MS) and fluorescence spectroscopy. Genes in the camalexin biosynthetic pathway were investigated by two-step reverse transcription polymerase chain reaction (PCR), GUS reporter gene assays and quantitative real time PCR (RT-qPCR).

Histones are the basic proteins complexed with DNA in the chromosomes of eukaryotic organisms. A previously undescribed histone (histone T.) was discovered in chromatin prepared from rainbow trout (Salmo gairdnerii) testes. Histone T was purified by selective extraction and ion-exchange chromatography on CM-cellulose. The homogeneity of this protein was examined by polyacrylamide disc gel electrophoresis, SDS disc gel electrophoresis, urea starch gel electrophoresis and gel filtration chromatography. Histone T was homogeneous as judged by the above different criteria. The molecular weight was found to be 14,500 by the mobility of formylated or acetylated histone Ton SDS gel electrophoresis. The amino acid composition and the N-terminal amino acid were determined. Peptide maps of the tryptic peptides of histone T and the major histones indicated that histone T is not a degradation product of the major histones. Details of the mechanism of initiation of protein synthesis in eukaryotes have only recently been discovered. Protamines are the small, highly basic proteins complexed with DNA in the mature sperm cells of most higher animals. The synthesis of protamine in trout testes was studied. Cell suspensions prepared from trout testes at the protamine stage of differentiation were incubated in vitro and were found to incorporate intact, isotopically labelled methionine into the N-terminal sequence, Met-Pro-Arg..., methionine residue is removed from protamine after chain completion. Enzymatic activity capable of cleaving the dipeptide, Met-Pro, was found in testis cell fractions. The amino group of methionine incorporated into ribosome-bound nascent protamine was not blocked. Methionine incorporation was extremely sensitive to inhibition by cyclo-heximide. The evidence obtained indicates a role for methionine in the initiation of protamine biosynthesis in the trout, a eukaryote / Medicine, Faculty of / Medical Genetics, Department of / Graduate

This thesis reports the results of studies on steroid metabolism in a subject with an adrenocortical carcinoma and hypoglycemia, in the testes obtained from a subject with virilizing male pseudohermaphroditism and in the testes of the normal rat. Incubations of cell-free homogenates of an adrenocortical carcinoma from a 51 year old female with severe hypoglycemia were performed using ³H-pregnenolone and ¹⁴C-progesterone as substrates. Transformation of ³H-pregnenolone to progesterone, dehydroepiandrosterone and androstenedione was observed; no metabolites of ¹⁴C-progesterone were detected. The excretion rate in urine of 3α ,17,21-trihydroxy-5β -pregnan-20-one, a metabolite of cortexolone, was elevated which suggests that a defect in 11β -hydroxylase activity was present. The excretion rates in urine of total 17-ketosteroids, 17-hydroxycorticoids and 17-ketogenic steroids were elevated; the excretion rates of testosterone, dehydroepiandrosterone, pregnandiol, pregnanetriol and free cortisol were not elevated. The etiology of the hypoglycemia that may accompany some adrenocortical tumors remains unknown. It was not possible to relate the results of the investigations of steroid metabolism reported herein to the hypoglycemia that was present. Steroid biosynthesis in vitro was investigated in testes obtained during puberty from a 14 year old subject with virilizing male pseudohermaphroditism. Cell-free homogenates of gonadal tissue efficiently metabolized ³H-pregnenolone, ¹⁴C-progesterone and ¹⁴C-androstenedione to testosterone; formation of estrone and estradiol-17β was not detected. 16α-Htdroxyprogesterone was formed from both ³H-pregnenolone and ¹⁴C-progesterone. The results are similar to those of others who have investigated the steroidogenic capacity of gonadal tissue in patients with male pseudohermaphroditism and feminization at puberty. A defect in the formation of progesterone from pregnenolone has been suggested to explain the results of a previous study in which the gonadal tissue obtained from a patient with virilizing male pseudohermaphroditism was incubated with ³H-pregnenolone as substrate. In the investigations reported herein, transformations of ³H-pregnenolone to testosterone and androstenedione occurred both via 17-hydroxypregnenolone and dehydroepiandrosterone and via progesterone and 17-hydroxyprogesterone. The failure of patients with virilizing male pseudohermaphroditism to masculinize during embryonic development contrasts with the virilization that occurs during puberty. A biochemical abnormality may exert a transient effect during embryonic development. Alternatively, the sensitivity to androgenic hormones may be subnormal in certain tissues and normal in other tissues of patients with virilizing male pseudohermaphroditism. The biosynthesis of testosterone from progesterone and pregnenolone was investigated in the rat testis. Time studies were performed using cell-free homogenates and ³H-progesterone and ¹⁴C-17-hydroxyprogesterone in combination as substrates. It was demonstrated that the side-chain cleavage of 17-hydroxyprogesterone is the rate-limiting reaction in the biosynthesis of testosterone from progesterone and the evidence suggested that 17-hydroxyprogesterone was present as a bound intermediate (at least in part). The progesterone 17-hydroxylase and the 17-hydroxyprogesterone side- chain cleavage enzyme of the rat testis can be solubilized by treatment of lyophilized microsomes with Triton N-101. Both enzymes displayed maximal activity at pH 6.8 and at 37°. Progesterone rather than pregnenolone is the preferred substrate for the 17α-hydroxylase. Either NADH or NADPH can serve as the reductant for active 17-hydroxylation of progesterone and for side-chain cleavage of 17-hydroxyprogesterone. The soluble fraction contains NADPH dehydrogenase, non-heme iron protein and cytochrome P-450. The presence of these compounds in association with the 17α-hydroxylase and the side-chain cleavage enzyme activities suggests that these reactions are catalyzed by elaborate enzymatic systems analogous to those required for 11β-hydroxylation and cholesterol side-chain cleavage in adrenal mitochondria / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate