First Mexican Consensus on Crohn's disease.

INTRODUCTION: Crohn's disease (CD) is a subtype of chronic and incurable inflammatory bowel disease. It can affect the entire gastrointestinal tract and its etiology is unknown. OBJECTIVE: The aim of this consensus was to establish the most relevant aspects related to definitions, diagnosis, follow-up, medical treatment, and surgical treatment of Crohn's disease in Mexico. MATERIAL AND METHODS: Mexican specialists in the areas of gastroenterology and inflammatory bowel disease were summoned. The consensus was divided into five modules, with 69 statements. Applying the Delphi panel method, the pre-meeting questions were sent to the participants, to be edited and weighted. At the face-to-face meeting, all the selected articles were shown, underlining their level of clinical evidence; all the statements were discussed, and a final vote was carried out, determining the percentage of agreement for each statement. RESULTS: The first Mexican consensus on Crohn's disease was produced, in which recommendations for definitions, classifications, diagnostic aspects, follow-up, medical treatment, and surgical treatment were established. CONCLUSIONS: Updated recommendations are provided that focus on definitions, classifications, diagnostic criteria, follow-up, and guidelines for conventional medical treatment, biologic therapy, and small molecule treatment, as well as surgical management.

The controlling nutritional status score: A promising tool for nutritional screening and predicting severity in ulcerative colitis patients. / La escala control nutricional (CONUT): una herramienta prometedora para el cribado nutricional y como predictor de severidad en la colitis ulcerosa crónica idiopática.

INTRODUCTION AND AIMS: The controlling nutritional status (CONUT) score has previously been shown to be useful for nutritional assessment and the prediction of several inflammatory and neoplastic diseases. The aim of the present study was to evaluate the potential use of the CONUT score as a method for nutritional screening and predicting severity in ulcerative colitis (UC). MATERIALS AND METHODS: The study was conducted on 60 patients diagnosed with UC. Demographic, clinical, and biochemical patient characteristics were collected from their clinical records, and disease severity was assessed using the Truelove and Witts scale (TWS). The risks for malnutrition were evaluated through the nutritional risk index and the CONUT score. RESULTS: More than 90% of the UC patients presented with malnutrition risk, according to the scores analyzed. Patients with a high (>6points) CONUT score presented with moderate-to-severe activity on the TWS. A higher CONUT score was also associated with an increase in C-reactive protein (CRP) (P=.002) and erythrocyte sedimentation rate (ESR) (P=.009). The data analysis was performed utilizing the SPSS version 19 program. CONCLUSIONS: The CONUT score could be a promising tool for evaluating nutritional status in UC patients and predicting UC severity.

Aspects of inflammatory bowel disease during the COVID-19 pandemic and general considerations. / Aspectos y consideraciones generales en la enfermedad inflamatoria intestinal durante la pandemia por COVID-19.

So far, available evidence suggests that patients with inflammatory bowel disease (IBD) are not at greater risk for developing COVID-19 infection. In regard to patients with IBD remission: 5-aminosalycylates (5-ASAs) do not increase the risk for infection and should be continued. There is no need to suspend them or lower the dose. Immunomodulating drugs, such as thiopurines and methotrexate, should be continued, without modifying doses (even in patients with positive SARS-CoV-2 infection). No type of biologic therapy should be suspended, unless there are signs of COVID-19. Regarding patients with IBD activity: the oral and/or topical 5-ASA dose should be optimized in cases of disease relapse. Budesonide MMX should be considered in cases of mild-to-moderate activity, to avoid systemic steroid use. Systemic steroids should be avoided whenever possible because doses above 20mg per day have an immunosuppressive effect, which could increase susceptibility to any type of infection, including COVID-19. The combined use of thiopurines with steroids and/or tumor necrosis factor (TNF) monoclonal antibodies should also be avoided because those combinations can increase the risk for infections, including COVID-19. Finally, biologic treatment with anti-TNF-alpha agents or any other mechanism of action, such as anti-integrins or anti-interleukins, should be suspended if patients become infected with SARS-CoV-2. The drugs can be restarted once the infectious process is resolved.

Auranofin-induced oxidative stress causes redistribution of the glutathione pool in Taenia crassiceps cysticerci.

Previously, we have studied the effect of the gold-compound auranofin (AF) on both thioredoxin-glutathione reductasa (TGR) activity and viability of Taenia crassiceps cysticerci. It was demonstrated that micromolar concentrations of AF were high enough to fully inhibit TGR and kill the parasites. In this work, the dynamics of changes in the glutathione pool of T. crassiceps cysticerci following the addition of AF, was analyzed. A dose-dependent decrease in the internal glutathione concentration, concomitant with an increase in ROS production was observed. These changes were simultaneous with the formation of glutathione-protein complexes and the export of glutathione disulfide (GSSG) to the culture medium. Incubation of cysticerci in the presence of both AF and N-acetyl cysteine (NAC) prevents all the above changes, maintaining cysticerci viability. By contrast, the presence of both AF and buthionine sulfoximine (BSO) resulted in a potentiation of the effects of the gold compound, jeopardizing cysticerci viability. These results suggest the lethal effect of AF on T. crassiceps cysticerci, observed at micromolar concentrations, can be explained as a consequence of major changes in the glutathione status, which results in a significant increase in the oxidative stress of the parasites.

Purification and characterization of Taenia crassiceps cysticerci thioredoxin: insight into thioredoxin-glutathione-reductase (TGR) substrate recognition.

Thioredoxin (Trx) is an oxidoreductase central to redox homeostasis in cells and is involved in the regulation of protein activity through thiol/disulfide exchanges. Based on these facts, our goal was to purify and characterize cytosolic thioredoxin from Taenia crassiceps cysticerci, as well as to study its behavior as a substrate of thioredoxin-glutathione reductase (TGR). The enzyme was purified >133-fold with a total yield of 9.7%. A molecular mass of 11.7kDa and a pI of 4.84 were measured. Native electrophoresis was used to identify the oxidized and reduced forms of the monomer as well as the presence of a homodimer. In addition to the catalytic site cysteines, cysticerci thioredoxin contains Cys28 and Cys65 residues conserved in previously sequenced cestode thioredoxins. The following kinetic parameters were obtained for the substrate of TGR: a Km of 3.1µM, a kcat of 10s(-1) and a catalytic efficiency of 3.2×10(6)M(-1)s(-1). The negative patch around the α3-helix of Trx is involved in the interaction with TGR and suggests variable specificity and catalytic efficiency of the reductase toward thioredoxins of different origins.

[Infection frequency in patients with chronic idiopathic ulcerative colitis]. / Frecuencia de infecciones en pacientes con colitis ulcerosa crónica idiopática.

BACKGROUND: Ulcerative Colitis (UC) is a chronic inflammatory bowel disease characterized by diffuse inflammation of the mucosa of the colon. Up to now, diverse observational studies have implicated a wide variety of pathogenic microorganisms as causal and exacerbating factors in UC. Clostridium difficile (C. difficile) infection has been associated with recurrence and treatment failure and its incidence in patients with UC has been on the rise in the last few years. AIMS: To determine the frequency of infection by different microorganisms in Mexican UC patients. PATIENTS AND METHODS: A total of 150 patients with definitive UC diagnosis were studied. All the stool tests for parasites and ova, stool cultures, tests for the C. difficile toxins A and B, and immunohistochemistry for Cytomegalovirus in colon segment biopsies were analyzed. Other demographic and clinical variables of the disease were recorded for their correlation with infection frequency. RESULTS: Infection frequency in UC patients was 28.00%. C. difficile infection was present in 0.013%. Other pathogens were found, such as Endolimax nana (9.00%), Entamoeba histolytica (3.00%), Cytomegalovirus (2.00%), Salmonella (2.00%), Shigella (0.70%), Toxoplasma gondii (0.70%) and Iodamoeba bütschlii (0.70%). CONCLUSIONS: Infection frequency was 28.00% in our study and C. difficile infection represented only 0.013%.

Purification, characterization and kinetic properties of the Taenia solium glutathione S-transferase isoform 26.5 kDa.

Glutathione S-transferases are major phase II detoxification enzymes. Taenia solium, a parasite of humans and pigs, is exposed to toxic products. The aim of this work was to purify and characterize a T. solium glutathione S-transferase isoform of 26.5 kDa (SGST26.5) in order to obtain its kinetic parameters. Homogeneous SGST26.5 was obtained by a simple purification procedure. SGST26.5 showed a p I of 7.07, and a native Mr of 60 kDa with 26.5 kDa subunits. The optimum activity for SGST26.5 was found at pH 6.5-7.0 in the range 10-42 degrees C. SGST26.5 had a specific enzyme activity of 78, 7.1, 6.6, and 0.7 microM min(-1) mg(-1) with CDNB, 1,2-dichloro-4-nitrobenzene, 2,4-hexadienal and trans-2-nonenal as substrates, respectively. It also had a kcat/ K(mCDNB)=2.15 x 10(3) M(-1 )s(-1), kcat/ KmGSH)=4.5 x 10(3) M(-1 )s(-1) and Vmax for GSH and CDNB=74 and 77 microM min(-1) mg(-1), respectively. SGST26.5 was inhibited in a noncompetitive form by cibacron blue, bromosulfophthalein and triphenyltin chloride. Inhibition studies as a function of inhibitor concentration show that the enzyme is a homodimer. Bireactant system analysis show that it follows an ordered sequential mechanism.

Unfolding kinetics of glutathione reductase from cyanobacterium Spirulina maxima.

The kinetics of the irreversible unfolding of glutathione reductase (NAD[P]H:GSSG oxidoreductase, EC 1.6.4.2.) from cyanobacterium Spirulina maxima was studied at pH 7.0 and room temperature. Denaturation was induced by guanidinium chloride and the changes in enzyme activity, aggregation state, and tertiary structure were monitored. No full reactivation of enzyme was obtained, even after very short incubation times in the presence of denaturant. Reactivation plots were complex, showing biphasic kinetics. A very fast early event in the denaturation pathway was the dissociation of tetrameric protein into reactivatable native-like dimers, followed by its conversion into a nonreactivatable intermediary, also dimeric. In the final step of the unfolding pathway the latter was dissociated into denatured monomers. Fluorescence measurements revealed that denaturation of S. maxima glutathione reductase is a slow process. Release of the prostethic group FAD was previous to the unfolding of the enzyme. No aggregated species were detected in the unfolding pathway, dismissing the aggregation of denatured polypeptide chains as the origin of irreversibility. Instead, the transition between the two dimeric intermediates is proposed as the cause of irreversibility in the denaturation of S. maxima glutathione reductase. A value of 106.6 +/- 3 kJ mol(-1) was obtained for the activation free energy of unfolding in the absence of denaturant. No evidence for the native monomer in the unfolding pathway was obtained which suggests that the dimeric nature of glutathione reductase is essential for the maintenance of the native subunit conformation.

Aggregation, dissociation and unfolding of glucose dehydrogenase during urea denaturation.

The effect of urea on glucose dehydrogenase from Bacillus megaterium has been studied by following changes in enzymatic activity, conformation and state of aggregation. It was found that the denaturation process involves several transitions. At very low urea concentrations (below 0.5 M), where the enzyme is fully active and tetrameric, there is a conformational change as monitored by an increase in intensity of the tryptophan fluorescence and a maximum exposure of organized hydrophobic surfaces as reported by the fluorescence of 4,4'-dianilino-1,1'-binaphthyl-5.5'-disulfonic acid. At slightly higher urea concentrations (0.75-2 M), a major conformational transition occurs, as monitored by circular dichroism and fluorescence measurements, in which the enzyme activity is completely lost and is concomitant with the formation of interacting intermediates that lead to a highly aggregated state. Increasing urea concentrations cause a complete dissociation to lead first a partially and eventually the complete unfolded monomer. These phenomena are fully reversible by dilution of denaturant. It is concluded that after urea denaturation, the folding/assembly pathway of glucose dehydrogenase occurs with the formation of intermediate species in which transient higher aggregates appear to be involved.

An alternative model for the transmembrane segments of the yeast H+-ATPase.

An alternative topological model for the yeast plasma membrane H(+)-ATPase from K. lactis was deduced by joint prediction, using 11 algorithms for the prediction of transmembrane segments complemented with hydrophobic moment analysis. Similarly to the model currently used in the literature, this alternative model contains 10 transmembrane segments, four in the N-half and six in the C-half of the protein. However, the distribution of the membrane-associated segments on the C-half of the enzyme differs in both models. Nine of the 10 transmembrane segments are highly hydrophobic with low hydrophobic moments, and are probably involved in structural roles. The fifth transmembrane segment is, on the other hand, less hydrophobic, with the highest hydrophobic moment, suggesting that this segment might have a dynamic role in the coupling of the hydrolysis of ATP with the translocation of protons across the membrane. The alignment of the Ca(2+)-ATPase, the Na(+)/K(+)-ATPase and the H(+)-ATPase sequences showed that these proteins have the same topology in the N-half, but important differences were found at the C-half of the enzymes. In contrast with the mammalian ATPases, the fifth transmembrane segment in the H(+)-ATPase appears early in the sequence, giving rise to a shorter cytoplasmic central loop. This alternative model will be useful in the designing of site-directed mutagenesis experiments and contains information for the fitting of the amino acid sequence into the transmembrane region of the three-dimensional model of the ATPase.

A novel ATP-diphosphohydrolase from human term placental mitochondria.

This report describes an ATP-diphosphohydrolase activity associated with the inner membrane of human term placental mitochondria. An enriched fraction containing 30 per cent of the total protein and 80 per cent of the total ATP-diphosphohydrolase activity was obtained from submitochondrial particles. ATP-diphosphohydrolase activity was characterized in this fraction. The enzyme had a pH optimum of 8 and catalysed the hydrolysis of triphospho- and diphosphonucleosides other than ATP or ADP. Pyrophosphate was also hydrolysed, but AMP or other monoester phosphates were not. The activity of ATP-diphosphohydrolase was dependent on Mg(2 + ), Ca(2 + )or Mn(2 + )and the enzyme substrate was the cation-nucleotide complex. An excess of free cation produced inhibition.ATP-diphosphohydrolase activity was stimulated at micromolar concentrations of calcium or magnesium in the presence of La-PPi. Negative cooperativity kinetics was observed with all substrates tested. The V(max)ranged from 150 to 300nmol of Pi released/mg/min. The [S](0.5)for nucleotides was 1-10m m and 182m m for PPi. The enzyme was inhibited by orthovanadate, but not by l -phenylalanine, oligomycin, sodium azide, P(1),P(5)-di(adenosine-5')pentaphosphate or sodium fluoride.The experimental evidence showing absence of inhibition by sodium azide and sodium fluoride, hydrolysis of pyrophosphate but not of monoester phosphates, and negative cooperativity suggested that this enzyme was a novel ATP-diphosphohydrolase.

Presence of two enzymes, different from the F1F0-ATPase, hydrolyzing nucleotides in human term placental mitochondria.

The hydrolysis of ATP, ADP or GTP was characterized in mitochondria and submitochondrial particles since a tightly-bound ATPase associated with the inner mitochondrial membrane from the human placenta has been described. Submitochondrial particles, which are basically inner membranes, were used to define the location of this enzyme. Mitochondria treated with trypsin and specific inhibitors were also used. The oxygen consumption stimulated by ATP or ADP was 100% inhibited in intact mitochondria by low concentrations of oligomycin (0.5 microgram/mg) or venturicidine (0.1 microgram/mg), while the hydrolysis of ATP or ADP was insensitive to higher concentrations of these inhibitors but it was inhibited by vanadate. Oligomycin or venturicidine showed a different inhibition pattern in intact mitochondria in relation to the hydrolysis of ATP, ADP or GTP. When submitochondrial particles were isolated from mitochondria incubated with oligomycin or venturicidine, no further inhibition of the nucleotide hydrolysis was observed, contrasting with the partial inhibition observed in the control. By incubating the placental mitochondria with trypsin, a large fraction of the hydrolysis of nucleotides was eliminated. In submitochondrial particles obtained from mitochondria treated with trypsin or trypsin plus oligomycin, the hydrolysis of ATP was 100% sensitive to oligomycin at low concentrations, resembling the oxygen consumption; however, this preparation still showed some ADP hydrolysis. Native gel electrophoresis showed two bands hydrolyzing ADP, suggesting at least two enzymes involved in the hydrolysis of nucleotides, besides the F1F0-ATPase. It is concluded that human placental mitochondria possesses ADPase and ATP-diphosphohydrolase activities (247).

Thermal denaturation of glutathione reductase from cyanobacterium Spirulina maxima.

The thermal unfolding of glutathione reductase (NAD[P]H:GSSG oxidoreductase EC 1.6.4.2.) from cyanobacterium Spirulina maxima was monitored by differential scanning calorimetry and circular dichroism at neutral pH. Covalent cross-linking of enzyme at different temperatures revealed dimer as the species undergoing the thermal transition. A single endotherm was observed, but its thermodynamic parameters showed dependence on the scan rate. In the transition zone, aggregation of the dimeric species was observed. Analysis of the enzyme heated at 80 degrees C revealed that the resultant species retained a high content of secondary structure. The addition of low concentrations of guanidinium hydrochloride resulted in a full cooperative thermal transition. A model in which the dimeric protein undergoes a partial unfolding in a kinetically controlled fashion is proposed, such that the experimental value of delta H(cal) results from the simultaneous occurrence of endothermic and exothermic events.

Detergent solubilization of 3 beta-hydroxysteroid dehydrogenase from dog pancreas.

Functional 3 beta-hydroxysteroid dehydrogenase coupled with isomerase (3 beta-HSD) was extracted from dog pancreatic mitochondria by treatment with the zwitterionic detergent CHAPSO. Increasing concentrations of this detergent led to a progressive and simultaneous solubilization of the pregnene (C-21) and androstene (C-19) dehydrogenase activities. Optimal solubilization of both C-21 and C-19 3 beta-HSD activities was achieved at a detergent/protein ratio of 0.6 (w/w). One hundred thirty percent of the initial particulate enzyme activities were recovered in the 105,000 g supernatant fluid with a 2.5-fold increase in the enzymatic specific activities. The C-21/C-19 activity ratios were 1.3 for mitochondria and 1.39 for the solubilized preparation. The apparent Km values for steroid substrates were unchanged after solubilization. Treatment of the mitochondrial suspension with sodium deoxycholate, CTAB, Lubrol XW, Brij 58, Emulgen 913 and Triton X-100 markedly decreased the 3 beta-HSD activities as a function of the detergent concentration and failed in to achieve solubilization.

Human placental estradiol 17 beta-dehydrogenase: structural and catalytic changes during urea denaturation.

The denaturation behavior of human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) in urea was studied by following changes in enzyme activity, conformation and oligomeric state. Results showed that the native --> unfolded transition follows a complex pattern, in which changes in both secondary and tertiary structure are simultaneous with changes in the aggregation state of enzyme. At relatively low urea (< 3 M), a major conformational transition, as monitored by CD and fluorescence measurements, is concomitant with an expanded state of the enzyme that coincides with its inactivation and the formation of polymeric species. Protein structural changes were also monitored by using the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid. The combined data suggest the existence of a molten globule state of dimeric enzyme promoted by low urea concentrations. Dilution of urea at this stage results in a full recovery of the enzymatic activity as well as of the native dimeric structure. Between 3 and 5 M urea estradiol 17 beta-dehydrogenase exists as a mixture of high molecular mass species which may be resolved by electrophoresis. In this range of urea concentration, only minor conformational changes were detected, although inactivation becomes to be irreversible. Above 5 M urea a second conformational transition takes place. Electrophoretic analysis of cross-linked samples revealed this stage results in the complete dissociation of enzyme toward unfolded monomer. It is concluded that the inactivation and unfolding of estradiol 17 beta-dehydrogenase during denaturation by urea occurs with the formation of intermediate species with different stability in which a molten globule-like state appears to be involved. The irreversibility of the process above urea 3 M is explained as the inability of aggregated enzyme to dissociate into native dimers.

Human placental 17 beta-hydroxysteroid dehydrogenase: secondary structure and circular dichroism demonstration of conformational changes upon NADP binding.

The secondary structure of human placental 17 beta-hydroxysteroid dehydrogenase in the absence and presence of NADP has been studied by circular dichroism spectroscopy. The conformational analysis of the NADP-containing enzyme shows that is an alpha/beta protein with 60% of regular secondary structure (38% of alpha helix, 22% of beta-strand structures), 20% of beta-turn and 20% of non-repetitive structure. These results were in good agreement with the information obtained using statistical and homology methods based on amino-acid sequence. On the other hand, 25% alpha-helix, 55% beta-strand, and 20% non-repetitive structure were estimated by circular dichroism for the cofactor-free enzyme. Addition of varying concentrations of NADP to the cofactor free enzyme is accompanied by circular dichroism spectral changes. From the variation in the magnitude of the positive band at 193 nm with increasing NADP concentration, a dissociation constant of 34 nM was obtained.

Denaturing behavior of glutathione reductase from cyanobacterium Spirulina maxima in guanidine hydrochloride.

The influence of guanidine hydrochloride (Gdn-HCl) on glutathione reductase from Spirulina maxima has been studied by measuring the changes in enzymatic activity, protein fluorescence, circular dichroism, thiol groups accessibility, and gel filtration chromatography. It was found that the denaturation process involves several intermediate states. At low, Gdn-HCl concentrations (Cm = 0.4 M), reductase activity was fully lost. However, below 3 M Gdn-HCl, this inhibition was freely reversible upon removal of the denaturing agent. Gel filtration experiments revealed that this reversible inhibition was not due to dissociation of the tetrameric enzyme. Structural studies strongly suggest that the conformation of this intermediate state is similar to that of native enzyme. A model in which a local region of the polypeptide chain assumes an extended conformation (D. T. Haynie, and E. Freire, Proteins 16,115-140) is proposed for the reversibly inactivated enzyme. Between 3 and 4 M Gdn-HCl (Cm = 3.5), the enzyme activity was irreversibly lost, this inhibition being concomitant with the loss of ellipticity, changes in both wavelength and intensity at the maximum of fluorescence emission, and dissociation of the enzyme into unfolded monomers; these results reveal that gross changes in the protein conformation occur under these conditions. At 4 M Gdn-HCl an equilibrium exists between the denatured forms of dimer and monomer, which is completely shifted toward the unfolded monomers at 5 M Gdn-HCl. Irreversibility in the Gdn-HCl-induced denaturation of S. maxima glutathione reductase was not due to aggregation of the unfolded enzyme.

Effect of inorganic phosphate on the self-associating properties of glutathione reductase from Spirulina maxima.

In the presence of millimolar concentrations of inorganic phosphate, native Spirulina maxima glutathione reductase (NAD[P]H:GSSG oxidoreductase EC 1.6.4.2.) changes its aggregation state. The oligomeric structure of the enzyme was notably dependent upon phosphate molarity, ranging from a dimer-tetramer equilibrium at relatively low phosphate concentrations into a tetramer-octamer equilibrium at moderate or high phosphate concentrations. In spite of the changes in quaternary structure, the tetramer remains as the most stable and abundant species. Sodium chloride solutions were not able to produce a similar effect, thus discarding an unspecific ionic strength effect.

Purification and characterization of glutathione reductase from Rhodospirillum rubrum.

Glutathione reductase (NAD(P)H:GSSG oxidoreductase EC 1.6.4.2.) was purified 1160-fold to homogeneity from the nonsulfurous purple bacteria Rhodospirillum rubrum (wild type). Specific activity of the pure preparation was 102 U/mg. The enzyme displayed a typical flavoprotein absorption spectrum with maxima at 274,365, and 459 nm and an absorbance ratio A280/A459 of 7.6. The amino acid analysis revealed an unusually high content of glycine and arginine residues. Titration of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) showed a total of two free thiol groups per subunit, one of which is made accessible only under denaturing conditions. An isoelectric point of 5.2 was found for the native enzyme. Km values, determined at pH 7.5, were 6.1 and 90 microM for NADPH and GSSG, respectively. NADH was about 2% as active as NADPH as an electron donor. The enzyme's second choice in disulfide substrate was the mixed disulfide of coenzyme A and glutathione, for which the specific activity and Km values were 5.1 U/mg and 3.4 mM, respectively. A native molecular weight of 118,000 was found, while denaturing electrophoresis gave a value of 54,400 per subunit, thus suggesting that R. rubrum glutathione reductase exists as a dimeric protein. Other physicochemical constants of the enzyme, such as Stokes radius (4.2 nm) and sedimentation coefficient (5.71 S), were also consistent with a particle of 110,000.

Delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in canine pancreas.

Activity of delta 5-3 beta-hydroxysteroid dehydrogenase coupled with steroid-delta 5-4-isomerase was demonstrated for the first time in the pancreas. The enzyme complex was assayed by measuring the conversion of pregnenolone to progesterone as well as of dehydroepiandrosterone to androstenedione and found to be localized primarily in the mitochondrial fraction of dog pancreas homogenates. The delta 5-3 beta-hydroxysteroid dehydrogenase used either NAD+ or NADP+ as co-substrates, although maximal activity was observed with NAD+. In phosphate buffer, pH 7.0 and 37 degrees C, the apparent Km values of the dehydrogenase were 6.54 +/- 0.7 microM for pregnenolone and 9.61 +/- 0.8 microM for NAD+. The apparent Vmax was determined as 0.82 +/- 0.02 nmol min-1 mg-1. Under the same conditions the Km values for dehydroepiandrosterone and NAD+ were 3.3 +/- 0.2 microM and 9.63 +/- 1.6 microM, respectively, and the apparent Vmax was 0.62 +/- 0.01 nmol min-1 mg-1.