Ipragliflozin as an Initial Therapy in Drug Naïve Subjects with Type 2 Diabetes.

OBJECTIVES: The aim of this study is to investigate ipragliflozin as an initial type 2 diabetes (T2DM) drug. METHODS: Ipragliflozin 25-50 mg/day monotherapy was performed with drug naïve subjects with T2DM (n=31). As a comparator, 12.5-25 mg/day alogliptin monotherapy was undertaken (n=32). At 3 months, levels of metabolic parameters were compared with those at baseline. FINDINGS: 4 subjects discontinued ipragliflozin due to intolerance or adverse events, while none dropped out with alogliptin. At 3 months, similar decreases of HbA1c levels were observed with these 2 drugs (10.21-8.31%, p<0.00001, with ipragliflozin, and 10.08-8.25%, p<0.00001, with alogliptin), however fasting blood glucose (FBG) levels decreased with significant inter-group differences (- 23.5% with iprgliflozin and - 10.8% with alogliptin). While similar increases of homeostasis model assessment (HOMA)-B levels were observed with these 2 drugs, HOMA-R levels significantly decreased only with ipragliflozin (-19.4%, p<0.02). Un-correlative link between HOMA-R and HOMA-B levels at baseline became significantly correlative (R=0.6017, p<0.001) only with ipragliflozin. Significant reductions of body mass index (BMI, -2.6%, P<0.05) were observed with ipragliflozin, however, no correlations between the changes of BMI and those of HbA1c or FBG were noted. CONCLUSIONS: These results suggest that ipragliflozin has good glycemic efficacy as an initial therapy in subjects with T2DM, although certain adverse events or tolerability issues are concerned. It improves insulin sensitivity and may restore the impaired beta-cell function. However body weight reduction with ipragliflozin is not associated with its glycemic efficacy.

Distinct glucose-lowering properties in good responders treated with sitagliptin and alogliptin.

BACKGROUND AND OBJECTIVES: All dipeptidyl peptidase 4 (DPP-4) inhibitors display similar glycemic efficacies, although they differ greatly in their chemical structures and pharmacological properties. This may be due to the inclusions of non- or poor-responders in the analysis, thereby masking the real effects of these drugs. The aim of this study was to identify any differences in diabetic parameters only in good responders treated with sitagliptin and alogliptin. METHODS: Treatment naïve subjects with type 2 diabetes mellitus were assigned to either sitagliptin 25-50 mg/day (n = 69) or alogliptin 12.5-25 mg/day monotherapy (n = 62) for 3 months. Only those who showed good response selected by a novel parameter called A1c index were further analysed (n = 24 for sitagliptin and n = 21 for alogliptin). RESULTS: At baseline, FBG and BMI were significantly higher and CPR-index was significantly lower in alogliptin good responders. At 3 months, while similar reductions of HbA1c were observed in these two groups, decreases of fasting blood glucose (FBG) were significantly higher in alogliptin good responders. Homeostasis model assessment (HOMA)-R significantly decreased only in alogliptin good responders, while HOMA-B similarly and significantly increased in these two groups. BMI significantly increased only in sitagliptin good responders. Significant negative correlations were observed between A1c index and changes (Δ) of HOMA-B in both groups. By contrast, significant positive and negative correlations were observed between ΔFBG and ΔHOMA-R, and between ΔFBG and ΔHOMA-B, respectively, only in alogliptin good responders. CONCLUSIONS: These results implicate that the effects on diabetic parameters and the glucose-lowering mechanisms of these two drugs might be different in those who have good response with these drugs. Accordingly, the choice of these drugs may be dependent on the characteristics of the patients.

Probable linagliptin-induced liver toxicity: a case report.

AIM: Unlike other dipeptidyl peptidase 4 (DPP-4) inhibitors, the excretion of linagliptin is mainly through a biliary route. Despite this fact, liver injury with linagliptin has thus far not been reported in the literature. However, this report describes the first case of probable linagliptin-induced liver toxicity. METHODS: The clinical history, diagnosis, investigations and drug treatment of the patient are reviewed here. RESULTS: A 58-year-old Japanese woman presented with fatigue, nausea, jaundice and marked elevations of hepatic enzymes 4weeks after starting linagliptin 5mg/day as monotherapy. No other medications were taken, and imaging studies revealed no other obvious causes of hepatic injury. Tests for viral serology and antinuclear antigen were negative. Symptoms disappeared and the levels of hepatic parameters (serum aminotransferases and biliary enzymes) slowly recovered after discontinuation of linagliptin. The slow recovery process may have been due to the very long half-life of the drug. The patient's Naranjo scale score was 6 and RUCAM score was 7. CONCLUSION: Although linagliptin currently carries no liver warnings, it may be necessary to monitor hepatic function in some patients upon administration of this drug until further evidence is obtained.

Functional interference between estrogen-related receptor alpha and peroxisome proliferator-activated receptor alpha/9-cis-retinoic acid receptor alpha heterodimer complex in the nuclear receptor response element-1 of the medium chain acyl-coenzyme A dehydrogenase gene.

We undertook a study of molecular interference of nuclear orphan receptors. Nuclear receptor response element-1 (NRRE-1) from the human medium-chain acyl coenzyme A dehydrogenase (MCAD) gene promoter was shown to contain three hexamer elements (site 1 through 3) that are known to interact with a number of nuclear receptors including chicken ovalbumin upstream promoter transcription factor (COUP-TF) and estrogen-related receptor alpha (ERRalpha). We demonstrated that the peroxisome proliferator-activated receptor alpha/9-cis-retinoic acid receptor alpha (PPARalpha/RXRalpha) heterodimer complex can also bind to the two hexamer repeat sequences (between site 1 and site 3) arranged as an everted imperfect repeat separated by 14 bp (ER14). Mutations of the putative core elements have shown that these three sites are differentially involved in ERRalpha and PPARalpha/RXRalpha binding. Homodimer of ERRalpha was shown to interact between site 1 and site 3 (ER14). To date, no nuclear receptor is known to bind to response elements over such long intervals. Interestingly, site 1 was shown to be essential for ERRalpha binding while site 3 supports its binding only in the presence of site 1. Furthermore, it was shown that the binding profile of ERRalpha and PPARalpha/RXRalpha are competitive rather than making a high order complex within NRRE-1. At the cellular level, transcriptional activation driven by the PPARalpha/RXRalpha complex was counteracted by the expression of ERRalpha in HeLa cells. These results suggest that ERRalpha and PPARalpha/RXRalpha could interfere with each other's function through binding to similar DNA elements, thereby finetuning the transcriptional outcome of the target gene. Our findings suggest a mechanism whereby multiple nuclear receptors can activate or repress DNA binding or transcription via a single pleiotropic regulatory element.

Transactivation of the IGFBP-2 promoter in human tumor cell lines.

Many cancers produce high amounts of the insulin-like growth factor binding protein (IGFBP)-2, which can influence the tumorigenicity and growth of tumor cells. In order to study the possible cause of elevated expression of IGFBP-2 in tumors, we investigated the transcriptional regulation by IGF of a 633-bp fragment of the human IGFBP-2 promoter in a transiently transfected choriocarcinoma (JAR) and a leukemic T-cell line (Molt-4) that express IGFBP-2 highly, and in a leukemic B-cell line (Raji) that expresses little IGFBP-2. Strong basal promoter activity, i.e. luciferase activity was measurable in all of the tumor cell lines. The introduction of equal amounts of normal IGF-I and IGF-II stimulated the transcription of IGFBP-2 only slightly. Synthetic IGF analogues with increased biological activity, however, caused a specific 2.0-3.3-fo1d transactivation of the promoter, as well as a 25% increase in IGFBP-2 mRNA. Synchronously, IGF analogues caused a decrease in the level of IGFBP-3 mRNA of about 45%, while the production of IGFBP-2 as measured by RIA increased in relation to IGFBP-3 by up to 15 times. Blocking with the IGF antagonist JB1 revealed partial involvement of the IGF-I receptor in the regulation of IGFBP-2 expression by locally produced IGF. We conclude, that the reduced ability of IGF analogues to form complexes with locally produced IGFBP may account for their increased biological activity in the stimulation of expression of IGFBP-2 and of cell growth. Since increased biological activity had also been demonstrated for natural pro-IGF forms often produced by tumors, pro-IGFs may be involved in the mechanism leading to elevated IGFBP-2 expression of tumors in vivo.

A putative white adipose tissue specific nuclear orphan receptor that interacts with the cAMP-response element of the human beta3-adrenergic receptor gene.

The authors previously reported that one of the cAMP-response elements (CREs) of the human beta3-AR gene, beta3CRE2, interacts with a nuclear factor which is distinct from CREB/ATF family. We named this factor WATSF-1 (white adipose tissue specific factor-1) since it is preferentially expressed in WAT. In this work, we have shown the absence of DNA binding or transcriptional activity of this factor in several non-adipose cells tested. By computer analysis, beta3CRE2 was found to constitute an octameric element that is highly homologous to the binding site for some members of the nuclear hormone receptor superfamily. Using the response elements of other adipocyte-specific nuclear receptors as competitors, a 'cross-talk' between WATSF-1 and these response elements has been demonstrated. However, the affinity of WATSF-1 for these response elements differs from that for beta3CRE2 (self), implying that WATSF-1 is distinct from these adipocyte-specific nuclear receptors. Furthermore the DNA-binding activity of WATSF-1 was shown to be enhanced by phosphatase treatment, suggesting that phosphorylation may play an important role in the functional modulation of this factor. In an effort to prove that it is indeed an adipocyte-specific factor, we used 3T3-L1 cells, a cellular model of WAT, that can undergo differentiation into adipocytes. The DNA binding and transcriptional activity of this factor appeared during differentiation of the cells. Taken together, these results demonstrate that WATSF-1 is a putative white adipocyte-specific nuclear orphan receptor induced during adipogenesis and is a transcriptional activator through one of the CREs of the human beta3-AR gene. Targeting this factor may be a novel therapeutic approach to stimulation of the beta3-AR signal transduction pathway in adipose tissues.

Hevin is down-regulated in many cancers and is a negative regulator of cell growth and proliferation.

We have cloned a human Hevin cDNA from omental adipose tissue of different patients by reverse transcription polymerase chain reaction and shown a sequence variation due to a possible polymorphism at amino acid position 161 (E/G). Hevin protein expressed in vitro showed molecular weights of approximately 75 kDa and 150 kDa, suggesting that Hevin may form a homodimer in vitro. Using Northern blots and a human expressed sequence tAg database analysis, Hevin was shown to be widely expressed in human normal or non-neoplastic diseased tissues with various levels. In contrast to this, its expression was strongly down-regulated in most neoplastic cells or tissues tested. However, neither the mechanism nor the physiological meaning of this down-regulation is known. As an initial step towards investigating the functional role of Hevin in cell growth and differentiation, we transiently or stably expressed this gene in cancer cells (HeLa 3S) that are devoid of endogenous Hevin and measured DNA synthesis (cell proliferation) by 5-bromo-2'-deoxyuridine incorporation. Hevin was shown to be a negative regulator of cell proliferation. Furthermore, we have shown that Hevin can inhibit progression of cells from G1 to S phase or prolong G1 phase. This is the first report which describes the function of Hevin in cell growth and proliferation. Through database analysis, Hevin was found to be located on chromosome 4 which contains loss of heterozygosity of many tumour suppressor genes. Taken together, these results suggest that Hevin may be a candidate for a tumour suppressor gene and a potential target for cancer diagnosis/therapy.

Identification and characterization of the putative retinoblastoma control element of the rat insulin-like growth factor binding protein-2 gene.

The authors previously identified a silencer of the rat IGFBP-2 gene. Sequence examination of the silencer has revealed that it contains the target sequence for the pRb (retinoblastoma) tumour suppressor gene, referred to as the retinoblastoma control element (RCE) which is frequently found in the regulatory element of cellular oncogenes and growth factors. The presence of RCE suggests that the IGFBP-2 gene may be regulated by the pRb tumour suppressor gene. An in vitro gel retardation assay has shown that the putative RCEs from the IGFBP-2 gene are complexed with multiple nuclear factors from the rat liver BRL-3A cells. These DNA-protein complexes were not detected with the nuclear extracts from the cells that were growth arrested at the G1/S border of the cell cycle by high cell density. Using specific antibodies, Sp1 was shown to be one of the components for the multiple DNA-protein complex while pRb does not appear to be directly involved in the formation of the complex.

Cell-density (cycle) dependent silencer of the rat insulin-like growth factor binding protein-2 (IGFBP-2) gene.

An element which has a negative effect on transcription has been identified in the 5'-flanking region of the rat insulin-like growth factor binding protein-2 (IGFBP-2) gene. This element was confirmed to be a silencer by truncation or by linking it to a heterologous promoter. The silencer activity disappeared in the growth-arrested BRL-3A cells at the G1/S border by high cell density where the IGFBP-2 production is highly elevated. This observation may represent a novel mechanism through which gene expression is controlled by modulation of a silencer. Taken together, these results suggest a regulatory link between cell growth and IGFBP-2 expression regulated by its silencer.

Quantification of the full length leptin receptor (OB-Rb) in human brown and white adipose tissue.

Levels of expression of the leptin receptor (OB-R) splice variants have been studied in human omental white and perirenal brown adipose tissues by reverse transcription-PCR. The level of mRNA expression of the full length form (OB-Rb) was approximately 15% of that of the sum of all splice variants in white or brown adipose tissue. In an attempt to quantify the gene expression of OB-Rb in human white adipose tissue, a quantitative competitive PCR technique was developed, using oligonucleotide primers designed for OB-Rb and an internal standard for a "MIMIC" competition strategy. The levels of expression of OB-Rb mRNA in the omental fat of lean and obese patients were compared and no difference could be observed between the two groups. The quantitative RT-PCR technique allows for a fast and accurate measurement of the expression of the OB-Rb mRNA in small tissue samples.

Interaction of nuclear factors with the cAMP response elements of the human ß(3)-adrenoceptor gene.

Four potential cyclic adenosine 3',5'-monophosphate (cAMP) response elements (CREs), each having at most two mismatches from the classical canonical sequence, have been identified in the 5'UTR of the human ß(3)-adrenoceptor gene by Liggett and Schwinn (1991). Recently, three of these CREs were shown to confer responsiveness to cAMP when cloned into a CAT reporter vector (Thomas et al., 1992). In this study, in vitro gel-retardation assays have shown that recombinant human CRE binding protein-1 (CREB-1) or activating transcription factor-1 (ATF-1) can interact specifically with these four putative CREs (termed ß(3)CRE2), although with different affinities. Nuclear extracts from human brown or white adipose tissue contain proteins interacting with ß(3)CRE3 and ß(3)CRE2. These adipose nuclear factors were shown by competition assays and the use of antibodies to differ from CREB-1 or ATF-1. The nuclear factor(s) interacting with ß(3)CRE2 was found in human and rat brown and white adipose tissues, but not in the other nonadipose tissues examined, i.e., rat lung, liver, kidney, and heart, suggesting an adipose tissue-specific DNA binding or expression pattern. ß(3)CRE2 is found to constitute a hexameric element that is highly homologous to the binding site for members of the nuclear hormone receptor superfamily, and a competition assay using this site has provided evidence that an adipose tissuespecific orphan member of this superfamily may bind to ß(3)CRE2. Reporter gene assays have indicated that ß(3)CRE2 and ß(3)CRE3 slightly repress the basal level of transcription and that ß(3)CRE2 confers cAMP responsiveness, whereas ß(3)CRE3 does not.

Cell-density-dependent modulation of the rat insulin-like-growth-factor-binding protein 2 and its gene.

The steady-state level of the rat insulin-like-growth-factor-binding protein 2 (IGFBP-2) and insulin-like-growth-factor-II (IGF-II) mRNA increased approximately 20-fold when BRL-3A cells were cultured at increasingly higher cell densities. This increase could not be accounted for by paracrine or autocrine factors, or by the addition of insulin, IGF-I, transforming growth factor beta (TGF-beta), cAMP or IGFBP-2 to the culture medium. A reporter gene assay carrying the promoter domain of the IGFBP-2 gene indicated that the promoter-dependent IGFBP-2 transcription is tenfold higher in high-density cells. The increase in the IGFBP-2 message was accompanied by an increase in the level of protein in the medium. When confluent BRL-3A cells were reseeded at low cell density, the IGFBP-2 mRNA disappeared at a rate significantly faster than in normal conditions. A protein synthesis inhibitor, cycloheximide, was able to prevent the decay of the message observed after the switch from high to low densities. In summary, these findings suggest a regulatory link between cell density and IGFBP-2.

Melatonin affects proopiomelanocortin gene expression in the immune organs of the rat.

The induction of opioid peptides derived from cells of the immune system is postulated to be the main mechanism involved in the immunomodulatory role of melatonin. In this study, it has been demonstrated for the first time that melatonin can act on the level of proopiomelanocortin (POMC) gene expression. The effect of the pineal hormone, administered in late-afternoon subcutaneous injections, was studied in the immune organs of adult male Wistar rats by means of a highly sensitive reverse transcription polymerase chain reaction method (RT-PCR), followed by polyacrylamide gel electrophoresis and densitometric analysis of the bands. It was demonstrated that melatonin stimulates the expression of the 3rd exon of the POMC gene in the lymph nodes and in bone marrow. No significant effects of the pineal hormone were observed in the spleen and thymus. The study establishes that the formation of short POMC transcripts in the bone marrow and lymph nodes may be upregulated by melatonin. Moreover, the pineal hormone exerts its effect without antigenic stimulation.

Genomic structure and regulation of the promoter of the rat insulin-like growth factor binding protein-2 gene.

We describe the complete genomic organization of the rat insulin-like growth factor binding protein-2 (rIGFBP-2) gene. This single-copy gene spans over 36 kilobases (kb) and is split into four exons of 475, 224, 141, and 472 nucleotides (nt), and three introns of 32 kb, 686, and 1793 nt, respectively. A single transcription start site (-90) was mapped by S1 protection assay and primer extension. The putative promoter of the rIGFBP-2 gene does not possess TATA or CAAT elements; however, it contains three GC-rich regions located 37, 57, and 81 nt 5' of the cap site. Deletion analysis of the 0.6-kb region of the upstream sequences and transfection of these constructs into BRL-3A and Chinese hamster ovary cells were used to localize possible cis-acting elements. The three GC boxes enhanced chloramphenicol acetyltransferase and luciferase transcription almost to the same level as the XbaI-NsphI (-579 to +1) fragment and displayed synergism and orientation dependence. In addition a similar positive effect on luciferase transcription has been obtained by cotransfecting these fragments with varying amounts of Sp1 expression vector into Drosophila cells that lack endogenous Sp1. In vitro gel mobility shift assays demonstrated that box 1 (GGGCGG), box 2 (GGGAGG), and box 3 (GGGAAGG) bind to SpI with variable affinities and display cooperativity. A protein that gave a similar DNA binding pattern was present in nuclear extracts of BRL-3A cells. To analysis using consensus or aberrant Sp1 elements and a polyclonal Sp1 antiserum to inhibit DNA binding were performed. These in vivo and in vitro data demonstrated that Sp1 plays an important role in the regulation of the expression of rIGFBP-2.

Sp1 interacts with the consensus sequence for Egr-1 gene product with a cellular factor(s) and activates the transcription through this element.

We report that Sp1 from nuclear extracts of BRL-3A cells interacts with the consensus DNA sequence for the Egr-1 gene product in an overlapping manner. Purified Sp1 failed to bind to this sequence and with the addition of sub-saturating level of nuclear extracts, the binding activity appeared. In Drosophila cells (SL2), exogenously expressed Sp1 activated the transcription through the Egr-1 site. These findings suggest that Sp1 can be targeted to a non-Sp1 (Egr-1) site with a cellular factor(s) and can activate the transcription through this element.

Functional interference between the ubiquitous and constitutive octamer transcription factor 1 (OTF-1) and the glucocorticoid receptor by direct protein-protein interaction involving the homeo subdomain of OTF-1.

The ubiquitous and constitutive octamer transcription factor OTF-1 (Oct 1) is the target of positive regulation by the potent herpes simplex virus trans-activator VP16, which forms a complex with the homeodomain of OTF-1. Here we present evidence that the glucocorticoid receptor can negatively regulate OTF-1 function by a mechanism that is independent of DNA binding. In vivo-expressed glucocorticoid receptor inhibited in a hormone-dependent manner activation of a minimal promoter construct carrying a functional octamer site. Moreover, expression of the receptor in vivo resulted in hormone-dependent repression of OTF-1-dependent DNA-binding activity in nuclear extract. In vitro, the DNA-binding activity of partially purified OTF-1 was repressed following incubation with purified glucocorticoid receptor. Cross-linking and immunoprecipitation experiments indicated that the functional interference may be due to a strong association between these two proteins in solution. Finally, preliminary evidence indicates that the homeo subdomain of OTF-1 that directs formation of a complex with VP16 may also be critical for interaction with the glucocorticoid receptor. Thus, OTF-1 is a target for both positive and negative regulation by protein-protein interaction. Moreover, the functional interference between OTF-1 and the glucocorticoid receptor represents a novel regulatory mechanism in the cross-coupling of signal transduction pathways of nuclear receptors and constitutive transcription factors.

High level expression of functional full length and truncated glucocorticoid receptor in Chinese hamster ovary cells. Demonstration of ligand-induced down-regulation of expressed receptor mRNA and protein.

Full length human glucocorticoid receptor and truncated receptor derivatives lacking the major amino-terminal trans-activating domain were expressed in stably transfected Chinese hamster ovary (CHO) cells. The receptors were co-expressed together with human metallothionein IIa, and the expression levels were amplified in the presence of increasing concentrations of metal. In amplified cells, both synthesized receptor forms showed the expected molecular weights, as assayed by affinity labeling and immunoblotting. They were expressed at concentrations of about 350,000-520,000 molecules/cell which corresponds to a 10-fold increase in receptor levels as compared to rat liver cells. The hormone (agonist or antagonist) binding properties of the expressed proteins were very similar to those characteristic of authentic glucocorticoid receptors in tissues or cultured cells. Moreover, the expressed proteins specifically recognized a glucocorticoid-response element sequence motif in in vitro protein-DNA binding experiments. The activation of a glucocorticoid-responsive reporter gene by the expressed full length receptor was dramatic (about 75-fold) and strictly ligand-dependent. In contrast, the expressed amino-terminal deletion mutant exhibited considerably weaker functional activity but showed normal hormone-binding properties. Upon exposure to dexamethasone in vivo, the expressed receptor mRNAs and proteins were down-regulated about 2- to 6-fold, indicating that regulatory signals important for autoregulation may be contained within structures corresponding to the ligand and DNA-binding domains. Transcription from the expression vector was not negatively regulated from the hormone, strongly arguing that receptor down-regulation was due to a post-transcriptional mechanism. In conclusion, this expression system should be a useful tool for further structural and functional studies of the receptor, including the biochemistry of its activation from a cryptic to a functional species, and its ligand-dependent autoregulation.

A cytochrome o-type oxidase of the thermophilic bacterium PS3 grown under air-limited conditions.

A cytochrome o-type oxidase from the thermophilic bacterium PS3 grown under air-limited conditions was purified by ion-exchange chromatography in the presence of a non-ionic detergent. The enzyme was composed of three subunits (60, 30, and 16 kDa) and seemed to contain two molecules of heme b as prosthetic groups. It contained no detectable copper. The reduced enzyme showed absorption bands at 426 and 558.5 nm, and a characteristic spectral change upon binding CO. It oxidized several cytochromes c and artificial dyes such as N,N,N',N'-tetramethyl-p-phenylenediamine and phenazonium methosulfate at appreciable rates. Its Km for O2 was low (0.09 microM). It was capable of transmembrane electron transfer, because when reconstituted into liposomes, it generated a membrane potential upon oxidation without pumping protons.

Cytochrome c-551 from the thermophilic bacterium PS3 grown under air-limited conditions.

A small-sized c-type cytochrome, designated cytochrome c-551, was prepared from membrane fraction of the thermophilic bacterium PS3 grown under air-limited conditions by extraction with cholate, precipitation with polyethylene glycol, and successive chromatographies with DEAE-cellulose and Sephacryl S-200 in the presence of a detergent. The purified sample contained approximately 1 mol of heme c per 10,000 g protein; it showed absorption bands at 551, 522 and 416 nm upon reduction, and a Soret peak at 409 nm upon oxidation. This cytochrome showed a single band of 10 kDa on polyacrylamide gel electrophoresis with sodium dodecyl sulfate. The isoelectric point of this cytochrome c-551 was pH 4.0. Cytochrome c-551 was suggested to play an important role in the respiratory chain with a terminal oxidase cytochrome o, which is produced under air-limited conditions, since cytochrome c-551 could mediate electron transfer between cytochrome bc1(b6f) complex and cytochrome o, showing quinol oxidase activity.

Quinol-cytochrome c oxidoreductase from the thermophilic bacterium PS3. Purification and properties of a cytochrome bc1(b6f) complex.

A quinol-cytochrome c oxidoreductase (cytochrome bc1 complex) has been purified from plasma membranes of a thermophilic Bacillus, PS3, by ion-exchange chromatography in the presence of Triton X-100. The purified enzyme shows absorption bands at 561-562 nm and 553 nm at room temperature, and 560, 551, and 547 nm at 80 K upon reduction, and gives an ESR signal similar to that of a Rieske-type iron sulfur center. Its contents of protohemes, heme c, and non-heme iron are about 23, 10, and 21 nmol/mg of protein, respectively. The enzyme consists of four polypeptides with molecular masses of 29, 23, 21, and 14 kDa judging from their electrophoretic mobilities in the presence of sodium lauryl sulfate. Since the staining intensities of the respective bands are almost proportional to their molecular masses, the monomer complex (87 kDa) of the subunits probably consists of a cytochrome b having two protohemes, a cytochrome c1 and an Fe2-S2-type iron sulfur center. The 29 and 21 kDa subunits were identified as cytochromes c1 and b, respectively, and the 23-kDa subunit is probably an iron-sulfur protein, since the 14-kDa polypeptide can be removed with 3 M urea without reducing the content of non-heme iron. Several characteristics of the subunits and chromophores indicate that the PS3 enzyme is rather similar to cytochrome b6f (a bc1 complex equivalent) of chloroplasts and Cyanobacteria. The PS3 complex catalyzes reduction of cytochrome c with various quinol compounds in the presence of P-lipids and menaquinone. The turnover number at pH 6.8 was about 5 s-1 at 40 degrees C and 50 s-1 at 60 degrees C. The enzyme is heat-stable up to 65 degrees C.