Intact Enzymes Research Articles

Trichoderma reesei has no true exo-cellulase: all intact and truncated cellulases produce new reducing end groups on cellulose

Adsorption to and formation of insoluble reducing end groups on cellulose was studied for intact enzymes and catalytic domains, ‘cores’, of the four major cellulases from Trichoderma reesei, CBH I, CBH II, EG I and EG III. Individual enzymes were incubated with NaBH 4-reduced, phosphoric acid swollen Avicel (regenerated cellulose) or with filter paper. Adsorption onto regenerated cellulose was rapid (equilibration reached within 2 min), but was slow onto filter paper (not completed after 24 h). On both substrates, less was bound of the core domains than of the intact enzymes. After reaching a maximum in adsorption, all the core domains except CBH I core were released again. In general, the desorption of the core enzymes was much faster than the rate of substrate conversion. All enzymes produced new reducing end groups on both substrates, and thus none of them is a true exo-cellulase. However, both the rate of formation and the amount was considerably higher for the EG enzyme than for the CBH's, which may justify the classification of cellulases into two groups, although the difference is quantitative rather than qualitative. EG III was the most endo-active of the enzymes, and CBH I the least.

Identification of topaquinone and its consensus sequence in copper amine oxidases.

The nature of the active site cofactor and the amino acid sequence flanking this structure have been determined in a range of copper amine oxidases. For enzymes from porcine plasma, porcine kidney, and pea seedlings, proteolytic digestion was performed on phenylhydrazone or p-nitrophenylhydrazone derivatives. Thermolysin treatment leads to relatively small active site peptides, which have been characterized by Edman degradation and by resonance Raman spectroscopy. Resonance Raman spectra of peptides show identical peak positions and intensities relative to each other and to a model p-nitrophenylhydrazone derivative of topaquinone hydantoin, establishing topaquinone as the cofactor in each instance. Edman degradation of peptides provides active site sequences for comparison to previous determinations with bovine serum and yeast amine oxidases. The available data establish a consensus sequence of Asn, Topa, Asp/Glu. Trypsin leads to significantly longer peptides, which reveal a high degree of sequence identity between plasma proteins from bovine and porcine sources (89%), with significantly decreased identity between the porcine serum and intracellular amine oxidases (56%). A lower degree of identity (45%) is observed between the pea seedling and mammalian enzymes. As an alternative to the isolation of active site peptides for topaquinone identification, visible spectra of intact proteins have been investigated. It is shown that p-nitrophenylhydrazone derivatives of native enzymes, active site-derived peptides, and a topaquinone model exhibit identical behavior, absorbing at 457-463 nm at neutral pH (pH 7.2) and at 575-587 nm in basic solution (1-2 M KOH). These spectral properties, which appear unique to topaquinone, provide a rapid and simple test for the presence of this cofactor in intact enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of axial ligand in the electronic structure of model compound I complexes

AbstractA quantum chemical study of the two low‐lying quartet states of seven model compound I iron–porphyrin complexes with varying axial ligands has been carried out using the INDO method. The varying axial ligands included in this study are five that are models for those in the intact enzymes: imidazole and imidazolate (model peroxidase HRP and CCP), CH3CONH2 (Gln175 mutant of CCP), PhO−1 (catalase), CH3S−1 (P450), and two that have been used in biomimetics of these enzymes: Cl−1 (hemin) and PhS−1 (model P450s). The purpose of these studies was to determine the role of the axial ligands in determining (i) the relative energies of the two nearly degenerate quartet electronic states of compound I, involved either as an a1u or a2u porphyrin π cation radical and (ii) the electron and spin distributions in the a1u and a2u radical cations of compound I. For most of the model complexes, including both HRP‐I and CAT‐I, a moderate effect of the axial ligand on the relative energy of these two states was observed and the a1u radical cation was found to be the ground state. The energy order of these two radical cations, however, was reversed in the P450‐I model complexes, indicating an association of the unique property of the FeO bond breaking with an a2u radical cation. The symmetry‐allowed overlap between the FeO and 3a2u orbitals may lower the activation energy for the FeO bond cleavage in P450‐I. However, the calculated electronic and spin properties, including the unpaired spin and net charge on the oxygen and the FeO bond overlap density, important determinants of the reactivity of this complex in the ligand–FeO region, are very similar for all complexes and in both cation states. © 1992 John Wiley & Sons, Inc.

Properties of subcloned subunits of bacterial acetohydroxy acid synthases.

The acetohydroxy acid synthase (AHAS) isozymes from enterobacteria are each composed of a large and small subunit in an alpha 2 beta 2 structure. It has been generally accepted that the large (ca. 60-kDa) subunits are catalytic, while the small ones are regulatory. In order to further characterize the roles of the subunits as well as the nature and the specificities of their interactions, we have constructed plasmids encoding the large or small subunits of isozymes AHAS I and AHAS III, each with limited remnants of the other peptide. The catalytic properties of the large subunits have been characterized and compared with those of extracts containing the intact enzyme or of purified enzymes. Antisera to the isolated subunits have been used in Western blot (immunoblot) analyses for qualitative and semiquantitative determinations of the presence of the polypeptides in extracts. The large subunits of AHAS isozymes I and III have lower activities than the intact enzymes: Vmax/Km is 20 to 50 times lower in both cases. However, for AHAS I, most of this difference is due to the raised Km of the large subunit alone, while for AHAS III, it is due to a lowered Vmax. The substrate specificities, R, of large subunits are close to those of the intact enzymes. The catalytic activity of the large subunits of AHAS I is dependent on flavin adenine dinucleotide (FAD), as is that of the intact enzyme, although the apparent affinities of the large subunits alone for FAD are 10-fold lower. Isolated subunits are insensitive to valine inhibition. Nearly all of the properties of the intact AHAS isozyme I or III can be reconstituted by mixing extracts containing the respective large and small subunits. The mixing of subunits from different enzymes does not lead to activation of the large subunits. It is concluded that the catalytic machinery of these AHAS isozymes is entirely contained within the large subunits. The small subunits are required, however, for specific stabilization of an active conformation of the large subunits as well as for value sensitivity.

The type II isoform of bovine brain protein L-isoaspartyl methyltransferase has an endoplasmic reticulum retention signal (...RDEL) at its C-terminus.

Bovine brain is known to contain two major isoforms of protein L-isoaspartyl methyltransferase (PIMT), an enzyme that facilitates repair of atypical L-isoaspartyl peptide bonds in proteins. Although the two isoforms can be separated by anion-exchange chromatography, they appear to have similar, if not identical, substrate specificities in vitro. The more basic type I isoform has been extensively characterized, and its complete sequence has been reported. The present study was undertaken in an attempt to understand the structural and functional uniqueness of the more acidic type II isoform. Electrospray mass spectrometry of the intact enzymes revealed that the type II isoform is approximately 43 amu heavier than the type I isoform. Cyanogen bromide cleavage followed by HPLC with on-line mass analysis revealed that the type II isoform contains a unique C-terminal fragment which is 43 amu heavier than the corresponding fragment from the type I isoform. Amino acid composition analysis and direct sequencing of this fragment indicate that the type II isoform ends in the sequence ...RDEL, while the type I is known to end in ...RWK. Since ...RDEL, like ...KDEL, serves as an effective endoplasmic reticulum retention signal, we propose that the type II isoform serves to repair damaged proteins within the endoplasmic reticulum or, perhaps, within some other specialized compartment of the cell. Comparison of the protein sequences of the two bovine brain isoforms to DNA sequences for rodent PIMT reported by others suggests that the type II isoform may be produced by splicing within the codon for Arg224.

Role of a carboxyl-terminal helix in the assembly, interchain interactions, and stability of aspartate transcarbamoylase.

The six individual catalytic polypeptide chains within the two catalytic trimers of Escherichia coli aspartate transcarbamoylase (ATCase; EC 2.1.3.2) are folded into two discrete structural domains interconnected in part by helix 12, which comprises residues 285-305 and is located near the carboxyl terminus of the chain. The essential role of this helix in folding of the chains and their assembly into ATCase was demonstrated by introducing a stop codon at the position corresponding to amino acid 284, 291, or 299. Cells containing these mutations are pyrimidine auxotrophs lacking ATCase-like protein in cell extracts. In contrast, stable active enzyme is formed from chains truncated at position 306 or 307, showing that all 310 amino acids are not required for assembly. Replacements of Gln-288, Asn-291, Arg-296, and Ala-298 were introduced to assess the effect of alterations within helix 12 on protein stability. Stability of the trimers was measured both by differential scanning microcalorimetry and by the rate of exchange of chains at 4 degrees C when mutant trimers were incubated with succinylated wild-type trimers. Melting temperatures of the mutant trimers spanned a range of more than 20 degrees C, with a few higher and others lower than that of wild-type trimers. Large changes in interchain interaction energies were observed for the trimers, but there was no direct correlation between the ease of dissociation of the trimers and their thermal stability. Calorimetry on the mutant holoenzymes revealed alterations in the interactions between trimers and regulatory subunits within the intact enzymes. The striking changes in stability of both trimers and holoenzymes demonstrated that effects of relatively localized amino acid replacements in helix 12 are manifested by indirect global alterations propagated throughout the structure.

Identification of a metalloproteinase co-purifying with rat tumour collagenase and the characteristics of fragments of both enzymes

A metalloproteinase similar or identical to stromelysin was shown to co-purify with interstitial collagenase from the rat mammary carcinoma cell line, BC1. The mixture of BC1 metalloproteinase and collagenase degraded casein, gelatin, fibronectin, fibrinogen, laminin, proteoglycan and type IV collagen, in addition to types I and II collagen. Using SDS-PAGE and zymography, the M 4 of both enzymes was 51·10 3. During storage, the 51·10 3 protein converted to fragments of M r 34·10 3 and 24·10 3, and isoelectric points of 4.6–5.3 and 5.7–6.0, respectively. The fragments were separated from the intact ( M r 51·10 3) enzymes by DEAE-Sepharose chromatography, but intact metalloproteinase and collagenase activities resisted separation by a range of chromatographic methods. The M r 34·10 3 fragment retained the proteinolytic activities of the intact enzymes, excepting collagenase cleavage of collagen types I and II. The M r 24·10 3 fragment had no proteinolytic activity, showed an increase in M r of 6·10 3 upon reduction, in common with the intact enzymes, and also had similar chromatographic properties to the intact enzymes. The data presented are consistent with a pattern of breakdown which is common to both collagenase and the metalloproteinase, and suggest that both enzymes are comprised of two protein domains.

Monoclonal antibodies to three phospholipase C isozymes from bovine brain.

Murine hybridoma cell lines secreting antibodies against the three bovine isozymes of phosphoinositide-specific phospholipase C (PLC) were established: 6, 23, and 12 lines were obtained for PLC-I (150 kDa), PLC-II (145 kDa), and PLC-III (85 kDa), respectively. The antibodies were purified from ascites fluid, and their properties were studied in detail. All the antibodies cross-reacted with their corresponding PLC enzymes, but not with the other two isozymes, suggesting that the three enzymes contain very different antigenic determinants. The six antibodies elicited by bovine PLC-I also cross-reacted with human and rat enzyme, whereas three each from anti-PLC-II antibodies and anti-PLC-III antibodies did not react with the enzymes from different species. Each antibody exerts different effects on the phosphatidylinositol-hydrolyzing activity of PLC. The most inhibitory antibody for either isozyme PLC-I or PLC-II exhibits 80% inhibition, whereas no more than 20% inhibition was observed for the anti-PLC-III antibodies. Purified PLC-I frequently contains catalytically active 140- and 100-kDa forms and an inactive 41-kDa protein in addition to the intact 150-kDa form, probably due to its high sensitivity to an unidentified endogenous protease. The five anti-PLC-I antibodies which bind to the denatured 150-kDa polypeptide also recognized the 140-kDa form, whereas only three cross-reacted with the 100-kDa form, and the remaining two bound to the 41-kDa protein. Competitive binding studies with intact PLC enzymes and Western blot experiments with proteolytic digests revealed that the 6 anti-PLC-I, 23 anti-PLC-II, and 12 anti-PLC-III antibodies bind at least five, six, and seven different epitopes on PLC-I, PLC-II, and PLC-III, respectively. The fact that these monoclonal antibodies bind to different epitopes on the same enzyme allowed one to develop a highly specific and sensitive tandem radioimmunoassay for quantitating PLC-I, PLC-II, and PLC-III. The principle of the assay is that binding of an 125I-labeled antibody to the antigen immobilized by another antibody at a distinctive binding site is proportional to the amount of antigen present. By using this method, PLC-I, PLC-II, and PLC-III could be measured quantitatively in the presence of other proteins, detergents, lipids, polyanions, and metal ions, all of which greatly affect the activity of PLC enzymes.

Studies of the cellulolytic system of Trichoderma reesei QM 9414. Analysis of domain function in two cellobiohydrolases by limited proteolysis.

Limited action of papain on the native forms of two cellobiohydrolases (CBH) from Trichoderma reesei (CBH I, 65 kDa, and CBH II, 58 kDa) leads to the isolation of the respective core fragments (56 kDa and 45 kDa) which are fully active on small, soluble substrates, but have a strongly reduced activity (respectively 10% and 50% of the initial value) on microcrystalline cellulose (Avicel). By partial sequencing at the C terminus of the CBH I core and at the N terminus of the CBH II core the papain cleavage sites have been assigned in the primary structures (at about residue 431 and 82 respectively). This limited action of papain on the native enzymes indicates the presence of hinge regions linking the core to these terminal glycopeptides. The latter conserved sequences appear either at the C or N terminus of several cellulolytic enzymes from Trichoderma reesei [Teeri et al. (1987) Gene 51, 43-52]. The specific activities of the intact enzymes and their cores on two forms of insoluble cellulose (crystalline, amorphous) differentiate the CBH I and CBH II in terms of adsorption and catalytic properties. Distinct functions can be attributed to the terminal peptides: for intact CBH II the N-terminal region contributes in the binding onto both cellulose types; the homologous C-terminal peptide in CBH I, however, only affects the interaction with microcrystalline cellulose. It could be inferred that CBH I and its core bind preferentially to crystalline regions. This seems to be corroborated by the results of CBH I/CBH II synergism experiments.

Subunit assembly and metabolic stability of E. coli RNA polymerase.

Immunological cross-reaction was employed for identification of proteolytic fragments of E. coli RNA polymerase generated both in vitro and in vivo. Several species of partially denatured but assembled RNA polymerase were isolated, which were composed of fragments of the two large subunits, beta and beta', and the two small and intact subunits, alpha and sigma. Comparison of the rate and pathway of proteolytic cleavage in vitro of unassembled subunits, subassemblies, and intact enzymes indicated that the susceptibility of RNA polymerase subunits to proteolytic degradation was dependent on the assembly state. Using this method, degradation in vivo was found for some, but not all, of the amber fragments of beta subunit in merodiploid cells carrying both wild-type and mutant rpoB genes. Although the RNA polymerase is a metabolically stable component in exponentially growing cells of E. coli, degradation of the full-sized subunits was found in two cases, i.e., several temperature-sensitive E. coli mutants with a defect in the assembly of RNA polymerase and the stationary-phase cells of a wild-type E. coli. The in vivo degradation of RNA polymerase was indicated to be initiated by alteration of the enzyme structure.

Antiproliferative monoclonal antibodies: detection and initial characterization.

Two monoclonal antibodies (MAB) are described which inhibit in vitro cellular proliferation in the absence of complement or effector cells. These MAB were produced by hybridomas made from mice immunized against human B lymphoma cells. The MAB were detected by using a colorimetric assay that quantifies proliferation based on the conversion of a yellow tetrazolium salt to a purple formazan product, a reaction that occurs only in metabolically active cells with intact mitochondrial enzymes. A human B lymphoblastoid cell was used as the screening target. RBC4 is an IgM MAB that modulates and immunoprecipitates the transferrin receptor. RBG5 is an IgG1 that binds to a nonmodulating cell surface determinant different from the transferrin receptor. Both MAB are active at low concentrations (RBC4, 0.5 microgram/ml and RBG5, 0.01 microgram/ml). Immunofluorescence staining of cell lines by RBC4 and RBG5 shows little correlation with inhibition by the antibodies. They differentially inhibit the proliferation of a panel of T, B, and myeloid cell lines. Both antibodies inhibit the proliferation of alloantigen or mitogen-activated human peripheral blood lymphocytes (PBL). Unstimulated PBL are not affected by either MAB. The RB MAB each cause different morphologic changes of target cells. Whereas RBC4-inhibited cells exhibit nonspecific changes, RBG5 causes a progressive increase in the size and nuclear number of a subset of inhibited cells.

Resolution of the flavocytochrome p-cresol methylhydroxylase into subunits and reconstitution of the enzyme.

An improved procedure is described for the isolation of the flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida as well as methods for the separation of its subunits in native form and their recombination to reconstitute the original flavocytochrome. Under appropriate conditions, the reconstitution is stoichiometric and results in complete recovery of the catalytic activity of the flavocytochrome. The separated flavoprotein subunit shows only 2% of the catalytic activity of the original enzyme on p-cresol and is characterized by converging lines in bisubstrate kinetic analysis, while the intact and reconstituted enzymes show parallel line kinetics in steady-state experiments. van't Hoff plots of the dependence of the dissociation constant of the subunits of PCMH on temperature show a break near 15 degrees C. Above this temperature, KD is characterized by a positive delta H value of 12.6 kcal mol-1; below 15 degrees C, the dissociation is essentially temperature independent. The subunit dissociation is strongly dependent on ionic strength in the oxidized form of PCMH but not in the reduced form of the enzyme. Reduction also lowers the KD significantly, while substrates and nonoxidizable competitive inhibitors lower the dissociation constant even further, suggesting a conformation change. Combination of the subunits to form PCMH entails a small but measurable change in the absorption spectra of the component proteins.

Lysosomal enzyme phosphorylation. Recognition of a protein-dependent determinant allows specific phosphorylation of oligosaccharides present on lysosomal enzymes.

We have investigated the basis for the specific recognition of lysosomal enzymes by UDP-GlcNAc:lysosomal enzyme N-acetylglucosaminylphosphotransferase. This enzyme is responsible for the selective phosphorylation of mannose residues on lysosomal enzymes. Two mammalian lysosomal enzymes, cathepsin D and uteroferrin, and two nonlysosomal glycoproteins were treated with endo-beta-N-acetylglucosaminidase H to remove those high mannose oligosaccharide units which are accessible on the native protein. These proteins were then tested as inhibitors of three different glycosyltransferases. The endo H-treated lysosomal enzymes were shown to be specific inhibitors of the phosphorylation of intact lysosomal enzymes. Proteolytic fragments of cathepsin D, including the entire light chain and heavy chain, did not retain the ability to be recognized by the N-acetylglucosaminylphosphotransferase. These findings indicate that the intact protein portion of lysosomal enzymes contains a specific recognition determinant which leads to high-affinity binding to the N-acetylglucosaminylphosphotransferase. The expression of this determinant appears to be dependent on the conformation of the protein.

Analysis of the catalytic domain of phosphotransferase activity of two avian sarcoma virus-transforming proteins.

Proteolytic digestion of the transforming protein of Rous sarcoma virus (pp60src) with trypsin, chymotrypsin, or thermolysin generated a 29,000-dalton fragment representing the carboxyl half of this molecule. This proteolytic fragment was able to phosphorylate pp60src-specific immunoglobulin as well as exogenous substrates such as angiotensin, casein, and tubulin. When quantitated on a molar basis, the protease-resistant fragment of pp60src had a greater specific activity than the intact enzyme. Digestion of pp90yes, the transforming protein of Y73 sarcoma virus with these proteases yielded a peptide of similar molecular weight which was capable of autophosphorylation as well as the phosphorylation of exogenous substrates. The proteolytic fragments of both pp60src and pp90yes displayed the same strict specificity for phosphorylation of tyrosine as the intact enzymes. These results indicate that the 29,000-dalton carboxyl end of pp60src and pp90yes can function independently as phosphotransferases and indicate that the catalytic domains of these molecules have a conformation which confers protection against limited conditions of proteolysis.

An aspartate transcarbamylase lacking catalytic subunit interactions. Study of conformational changes by ultraviolet absorbance and circular dichroism spectroscopy.

A modified form of aspartate transcarbamylase is synthesized by Escherichia coli in the presence of 2-thiouracil which does not exhibit homotropic cooperative interactions between active sites yet retains heterotropic cooperative interactions due to nucleotide binding. The conformational changes induced in the modified enzyme by the binding of different ligands (substrates, substrate analogs, a transition state analog, and nucleotide effectors) were studied using ultraviolet absorbance and circular dichroism difference spectroscopy. Comparison of the results for the modified enzyme and its isolated subunits to those for the native enzyme and its isolated subunits showed that the conformational changes detected by these methods are qualitatively similar in the two enzymes. Comparison of the absorbance difference spectra due to the binding of a transition substrate analog to the intact native or modified enzymes to the corresponding results for the isolated subunits suggested that ligand binding causes an increased exposure to solvent of certain tyrosyl and phenylalanyl residues in the intact enzymes but not in the isolated subunits. This result is consistent with a diminution of subunit contacts due to substrate binding in the course of homotropic interactions in the native enzyme. Such conformational changes, though perhaps necessary for homotropic cooperativity, are not sufficient to cause homotropic cooperativity since the modified enzyme gave identical perturbations. Interactions of the transition state analog, N-(phosphonacetyl)-L-aspartate, with the modified enzyme were studied. Enzyme kinetic data obtained at low aspartate concentrations showed that this transition state analog does not stimulate activity, but rather exhibits the inhibition predicted for the total absence of homotropic cooperative interactions in the modified enzyme. Spectrophotometric titrations of the number of catalytic sites with the transition state analog showed that the modified enzyme and its isolated subunits possess, respectively, four and two high affinity sites for the inhibitor instead of six and three observed in the case of the normal enzyme and its isolated catalytic subunits. These results are correlated with the lower specific enzymatic activities of the modified enzyme and its catalytic subunits compared to the normal corresponding enzymatic species.

Bioenergetics in clinical medicine: prevention by forms of coenzyme Q of the inhibition by adriamycin of coenzyme Q10-enzymes in mitochondria of the myocardium.

Adriamycin inhibits the succinoxidase system and the NADH-oxidase system. Both of the intact mitocondrial enzymes and the pentane-extracted preparations are inhibited. The inhibition can be prevented by a molar ratio of coenzyme to adriamycin of 3:1 for coenzyme Q10 (ubiquinone), 5:1 for coenzyme Q7, and 5:1 for coenzyme Q4. Prevention of inhibition was observed in the decreasing order of coenzyme Q10 greater than coenzyme Q7 greater than H6 coenzyme Q4 greater than coenzyme Q4. Adriamycinone was three times more inhibitory than adriamycin, which is compatible with a less polar fragment necessary to inhibit the lipoidal coenzyme Q10. Daunomycinone was not inhibitory at a concentration at which adriamycinone is effective, indicating that the hydroxyl group of the latter could be binding at the receptor, since it should not influence electron transfer or rings B and C.

Baker's yeast flavocytochrome b2 (L-(+)-lactate dehydrogenase). An immunological study of structure and function.

Baker's yeast flavocytochrome b2 has been suggested before to be a polyglobular protein formed after a gene fusion process. It is known that one of the potential globules, the heme-binding core, can exist in the absence of the rest of the protein. Using antibodies elicited against the whole enzyme and against the core, we show in this paper that part of the core surface, and in particular the mouth of the heme-binding crevice, must be exposed in the complete enzyme molecule. Antibodies inhibit the activity of intact and proteolytically-cleaved enzymes, which normally show a number of differences in some kinetic parameters. Interestingly, antibodies against the core induce a modification of some kinetic constants for the cleaved enzyme (in particular the Km for the substrate and Ki for D-(-)-lactate, bringing them back to values similar to those for the intact enzyme. These results can be interpreted as a tightening of the cleaved enzyme by anti-core antibodies. The conformational effect is transmitted from the heme-binding region to other parts of the molecule. This implies some intimate contacts between the core and the rest of the protein.

Enteropancreatic circulation of digestive enzymes.

Intact digestive enzymes can be absorbed by the intestine and resecreted by the pancreas. The pancreas, therefore, appears to be able to recycle proteins much as the liver recycles bile salts, although the magnitude of this process remains uncertain.

Persistent postpubertal elevation of activity of steroid 5alpha-reductase in the adrenal of rat pseudohermaphrodites and correction by large doses of testosterone or DHT.

The development of adrenal androgen metabolizing enzymes with androstenedione and testosterone as substrates has been studied in the rat pseudohermaphrodite and its King-X Holtzman male littermates and females. Enzymatic activity has been determined by the separation and quantitation of the products formed from the labeled androgen substrates by a partition system on thin-layer chromatography, and by trimethylsilylether derivative formation and radio-gas-liquid chromatography. The separations have been confirmed by recrystallization. Serum corticosterone is significantly higher in the pseudohermaphrodite than in males or females indicating intact corticoidogenic enzymes. The development of adrenal androgen metabolizing enzymes in the pseudohermaphrodite differs from that of its littermate male primarily by a persistent postpubertal elevation of activity of 5α-reductase and a failure of a postpubertal rise in 17-ketoreductase, the enzyme converting the 17-ketone of androstenedione to the 17β-alcohol of tes...

Stability of oxidative phosphorylation and structural changes of mitochondria in ischemic rat liver

Mitochondria isolated from rat livers ischemic at 24 ° for up to 13 hr exhibited nearly normal capacity to phosphorylate added ADP ( ADP 0 ratios) in the presence of bovine serum albumin (BSA). Respiratory control ratios (rate with ADP/rate without ADP) remained nearly normal for the first 3 hr, then declined somewhat. However, even at 13 hr the ratios were still 2 to 3 compared to normal values of 4 to 6. The decline of respiratory control was due primarily to a decline in the State 3 respiratory rate (i.e., with ADP and substrate). The demonstration of relatively normal oxidative phosphorylation in mitochondria isolated from livers after prolonged periods of ischemia at 24 ° is dependent on the use of BSA in the isolation medium and/or in the test medium. In the complete absence of BSA, or at 37 ° in the presence of BSA, the respiratory control is lost rapidly. These data are compatible with release of fatty acids in the ischemic cell being responsible for the apparent damage to mitochondria. Electron microscopic studies show very slow changes with time in mitochondria in ischemic liver cells, greater changes in the isolated mitochondria. During the isolation procedures and in the test medium, binding by BSA can remove the fatty acids and their uncoupling and inhibitory effects, revealing relatively intact enzymes of oxidative phosphorylation.