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Biomedical subjects

M C Liu

Publications and source records attributed to M C Liu.

At least 163 records · Page 9Linked to original sources
PubMed1991

Purification and characterization of a membrane-bound tyrosine-O-sulfate-binding protein from bovine liver.

A membrane-bound 175-kDa tyrosine-O-sulfate (TyrS)-binding protein from bovine liver was purified to electrophoretic homogeneity. The purified protein exhibited an apparent molecular weight of 175,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Upon SDS-PAGE under nonreducing conditions, the TyrS-binding protein migrated considerably faster than the dimeric fibronectin, indicating its presence in the monomeric form and therefore the absence of a disulfide-bonded subunit structure. The purified TyrS-binding protein was found to bind concanavalin A-Sepharose and yielded a positive reaction toward periodic acid-Schiff (PAS) staining, indicating its glycoprotein nature. The purified TyrS-binding protein displayed strong binding to TyrS, but not the unmodified tyrosine, covalently bonded to Sepharose. Using a tyrosine-sulfated cholecystokinin octapeptide (CCK-8) as the ligand in a radioimmunoassay, it was found that the binding of the TyrS-binding protein was pH-dependent, being strong from pH 8.0 down through 6.5, and becoming dramatically weaker at pH below 6.0. Divalent cations, added in the assay mixture, exerted significant promoting effects on the binding in the order Mn+2 greater than Ca2+ greater than Mg2+. Western blot analysis clearly showed that the purified TyrS-binding protein was capable of forming complexes with two tyrosine-sulfated proteins, fibronectin and fibrinogen, but not two non-tyrosine-sulfated proteins, transferrin and albumin. These results provided support to a role of the TyrS-binding protein being a putative receptor for tyrosine-sulfated proteins.

Amino Acids↗
PubMed1991

Identification of complexes between the tyrosine-O-sulphate-binding protein and tyrosine-sulphated proteins in bovine liver membrane lysates.

Rabbit antiserum against electrophoretically purified bovine liver tyrosine-O-sulphate (TyrS)-binding protein was prepared. Affinity-purified antibodies from the antiserum were found to be capable of immunoprecipitating the TyrS-binding protein from the sodium choleate extract of a bovine liver microsomal membrane fraction. Using purified specific antibodies as the probe, Western blot analysis for the presence of TyrS-binding protein/tyrosine-sulphated protein complexes in bovine liver membrane lysates was performed. It was found that the TyrS-binding protein co-precipitated with three tyrosine-sulphated proteins (fibronectin, fibrinogen and complement C4) immunoprecipitated by their respective antibodies. In contrast, for the two non-tyrosine-sulphated proteins (haptoglobin and transferrin) tested, co-precipitation of the TyrS-binding protein was not observed. On employing an affinity gel fractionation technique, it was shown that partially purified TyrS-binding protein exhibited binding affinity towards Sepharose gels covalently bonded to fibronectin or fibrinogen, but not towards Sepharose gels bonded to albumin or transferrin. These results indicate that the TyrS-binding protein formed complexes with tyrosine-sulphated proteins both in vivo and in vitro, and thus provide support for the putative role of the former being the receptor of the latter.

Animals↗
PubMed1991

Synthesis and anticancer activity of various 3'-deoxy pyrimidine nucleoside analogues and crystal structure of 1-(3-deoxy-beta-D-threo-pentofuranosyl)cytosine.

Various 3'-deoxy pyrimidine nucleoside analogues have been synthesized for evaluation as potential anticancer and antiviral agents. Among these compounds, 1-(3-deoxy-beta-D-threo-pentofuranosyl)cytosine (10, 3'-deoxy-ara-C) and 3'-deoxycytidine (22) had significant anticancer activity against CCRF-CEM, L1210, P388, and S-180 cancer cell lines in vitro, producing ED50 values of 2, 10, 5, and 34 microM, respectively, for 3'-deoxy-ara-C (10); and 25, 5, 2.5, and 15 microM, respectively, for 3'-deoxycytidine (22). Thus, 3'-deoxy-ara-C (10) was 12.5 times more active against CCRF-CEM cells than 3'-deoxycytidine (22). The 2'-O-acetyl, 5'-O-acetyl, and 2',5'-di-O-acetyl derivatives of 3'-deoxy-ara-C (10), compounds 34, 31, and 30, demonstrated anticancer activity in the same range as 3'-deoxy-ara-C (10) against CCRF-CEM, L1210, P388, and S-180 cells. The 5'-O-acetyl derivative (31) had significantly greater activity against CCRF-CEM with an ED50 value of 0.4, but this compound also showed similar activity, as did 3'-deoxy-ara-C, against L1210, P388, and S-180 with ED50 values of 3, 3, and 13 microM, respectively. 3'-Deoxy-ara-C was also evaluated in vitro against HSV-2, HCMV, and GPCMV viruses and was found to be not very active with respective IC50 values of 110, 220, and 1000 microM. The single-crystal structure of 3'-deoxy-ara-C (10) was determined by X-ray crystallography. There are two molecules of the nucleoside and one molecule of water in the asymmetric unit. The sugar moieties of the two nucleoside molecules adopt different conformations. In molecule A, the ring pucker is C3'-endo with P = 18.7 degrees and tau m = 37.3 degrees, while the CH2OH side chain is gauche+. In molecule B, the ring pucker is C2'-endo with P = 156.8 degrees and tau m = 37.8 degrees and the side chain is trans.

Animals↗
PubMed1991

Synthesis and anticancer and antiviral activities of various 2'- and 3'-methylidene-substituted nucleoside analogues and crystal structure of 2'-deoxy-2'-methylidenecytidine hydrochloride.

Various 2'- and 3'-methylidene-substituted nucleoside analogues have been synthesized and evaluated as potential anticancer and/or antiviral agents. Among these compounds, 2'-deoxy-2'-methylidene-5-fluorocytidine (22) and 2'-deoxy-2'-methylidenecytidine (23) not only demonstrated potent anticancer activity in culture against murine L1210 and P388 leukemias, Sarcoma 180, and human CCRF-CEM lymphoblastic leukemia, producing ED50 values of 1.2 and 0.3 microM, 0.6 and 0.4 microM, 1.5 and 1.5 microM, and 0.05 and 0.03 microM, respectively, but also were active in mice against murine L1210 leukemia. Of all the tested drug dosage levels (25, 50, and 75 mg/kg, respectively) compound 23 had no toxic deaths and compound 22 yielded only one toxic death at the highest dosage level. On the contrary, in the same study, 1-beta-D-arabinofuranosylcytosine (ara-C) resulted in 2/5, 5/5, and 5/5 toxic deaths, respectively. Both compounds 22 and 23 have shown better anticancer activity than ara-C, yielding higher T/C x 100 values and some long-term survivors (greater than 60 days). In addition, compounds 22 and 23 were found to have, respectively, approximately 130 and 40 times lower binding affinity for cytidine/deoxycytidine deaminase derived from human KB cells compared to ara-C, suggesting that the two 2'-methylidene-substituted analogues may be more resistant to deamination. Cytoplasmic deoxycytidine kinase (dCK) was required for compounds 22 and 23 action. Furthermore, compounds 14, 22, 23, and 24 also have antiherpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) activity in cell culture. In addition, the crystal structure of 2'-deoxy-2'-methylidenecytidine hydrochloride (23-HCl) was determined by X-ray crystallography.

Animals↗
PubMed1991

Profile of prostanoid release following antigen challenge in vivo in the skin of man.

We have previously characterized the kinetics of prostaglandin D2 (PGD2) production at cutaneous sites of allergic inflammation employing a blister-chamber model. In this study, a more complete profile of prostaglandins released in vivo was obtained. PGD2 release, as measured by radioimmunoassay and by combined gas chromatography-mass spectrometry, was evident within 1 h after antigen challenge with maximal levels occurring 3-4 h post-challenge. The 11-ketoreductase metabolite of PGD2, 9 alpha, 11 beta-prostaglandin F2 was present in blister fluid from three of six patients at the time of maximal levels of PGD2. The stable non-enzymatic hydrolysis product of prostacyclin, 6-keto-prostaglandin F1 alpha, was significantly elevated in blister fluid from five of six patients following antigen challenge. In these subjects, the levels of 6 kappa-PGF1 alpha were highest in samples obtained 1 and 2 h after antigen challenge and remained significantly elevated until 5 h post-challenge. Levels of prostaglandin E2, prostaglandin F2 alpha and thromboxane B2 did not vary significantly. These studies suggest that following antigen challenge two fatty-acid cyclo-oxygenase products of arachidonic acid are released, PGD2 and prostacyclin. The 11-ketoreductase metabolism of PGD2 to 9 alpha, 11 beta-PGF2 could represent a mechanism by which the biological effects of PGD2 are prolonged in cutaneous tissue. The presence of 6 kappa-PGF1 alpha in the blister fluid suggests that significant prostacyclin release occurs as the result of antigen challenge and could represent a mechanism by which the prolonged microvascular response in cutaneous tissue may occur.

6-Ketoprostaglandin F1 alpha↗
PubMed1991

Immediate and late inflammatory responses to ragweed antigen challenge of the peripheral airways in allergic asthmatics. Cellular, mediator, and permeability changes.

Asthma may represent the clinical manifestations of a unique form of chronic airway inflammation and is often associated with allergy. To better define the components of allergic inflammation in the lung, fluids obtained by bronchoalveolar lavage (BAL) were examined for cells, inflammatory mediators, and markers of airway permeability 5 min and 19 h following instillation of ragweed antigen directly into an airway segment of allergic asthmatic subjects. The 5-min response to antigen challenge (n = 10) was characterized by 17- to 208-fold increases in histamine, prostaglandin D2 (PGD2), and its metabolite, 9 alpha,11 beta-PGF2, thromboxane B2, and 6-keto-PGF1 alpha compared with a saline-challenged segment (0.004 less than p less than 0.017). The increases in most of these mediators were significantly correlated with each other (0.0001 less than p less than or equal to 0.01), and the magnitude of all significant mediator increases was directly correlated with skin test sensitivity to ragweed antigen (0.007 less than or equal to p less than or equal to 0.05). There was also a slight increase in kinins (p = 0.04). Changes in cells and airway permeability were not detected. In contrast, the 19-h response to antigen challenge (n = 9) was characterized by a 13-fold increase in total cells recovered by BAL. Eosinophils, basophils, and lymphocytes were significantly increased and comprised 38, 1, and 9% of total cells, respectively. A neutrophil influx was also observed but was not specific for antigen challenge since a similar change was observed in a sham, saline-challenged site.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Purification and characterization of the apically secreted 80 KDa glycoprotein from Madin-Darby canine kidney (MDCK) cells.

An 80 KDa glycoprotein (gp 80), known to be released predominantly from the apical surface by filter-grown Madin-Darby canine kidney cells, was purified to electrophoretic homogeneity. Purified gp 80 was found to have a disulfide-bonded dimeric structure, and appeared to exist in two molecular forms, a major (high-molecular weight) form consisting of a 46 KDa subunit and a 39 KDa subunit and a minor (low-molecular weight) form consisting of a 46 KDa subunit and a 33 KDa subunit. Upon de-glycosylation by N-glycanase treatment, the 46 KDa subunit was converted to a 25.6 KDa form, whereas both the 39 KDa and the 33 KDa subunit gave rise to a 21.1 KDa form. V8 protease mapping of deglycosylated polypeptides revealed the 39 KDa and the 33 KDa subunit to have nearly identical band patterns, which also exhibited a high degree of homology to that derived from the 46 KDa subunit. Radioimmunoassays revealed that the binding of the purified gp 80 to fibrinogen (or heparin) was dependent on both pH and divalent cations. Furthermore, binding of gp 80 to immobilized fibrinogen (or heparin) was inhibited in the presence of free fibrinogen (or heparin) added in the assay mixture.

Amidohydrolases↗
PubMed1990

Metabolic activation of 4-ipomeanol in human lung, primary pulmonary carcinomas, and established human pulmonary carcinoma cell lines.

4-Ipomeanol (IPO) is a pulmonary-specific toxin that is metabolically activated by a cytochrome P450 pathway in lung tissue. In this study, IPO metabolism, as determined by measurement of [14C]IPO covalent binding, was evaluated in a diverse sampling of 18 established, human lung cancer cell lines as well as in normal lung tissue and primary lung carcinoma tissue obtained at the time of thoracotomy from 56 patients with lung cancer. [14C]IPO covalent binding in lung cancer cell lines ranged from 248 to 1,047 pmol of bound [14C]IPO per milligram of protein per 30 minutes (mean +/- SE = 547 +/- 62.2). IPO metabolism in normal lung tissue ranged from 12 to 2,007 pmol of covalently bound [14C]IPO per milligram of protein per 30 minutes (mean +/- SE = 549 +/- 60). In lung cancer tissue, values ranged from 0 to 2.566 pmol of covalently bound [14C]IPO per milligram of protein per 30 minutes (mean +/- SE = 547 +/- 60, P greater than .3). When patients were divided into smokers and current non-smokers (no tobacco products smoked for greater than 6 mo), no effects of cigarette smoking were observed for either normal lung tissue or lung tumor tissue (P greater than .1 in all instances). A wide range of IPO metabolic activity was observed among different histological classifications of lung cancer cell lines and of fresh lung cancer tissues. IPO metabolism was simultaneously compared in normal lung tissue and lung cancer tissue from individual patients, but no positive correlation was observed (r = .10; P greater than .30). The results clearly demonstrate a wide range of IPO metabolism in both normal and lung cancer cells and indicate that a wide diversity of human lung cancers possess the metabolic enzyme system(s) necessary for the bioactivation of IPO to a potentially cytotoxic intermediate. Therefore, the continued exploration for any possible therapeutic potential of IPO in patients with lung cancer appears warranted.

Biotransformation↗
PubMed1990

Expression of CYP1A1 gene in patients with lung cancer: evidence for cigarette smoke-induced gene expression in normal lung tissue and for altered gene regulation in primary pulmonary carcinomas.

The major polycyclic aromatic hydrocarbon inducible-cytochrome P4501A1 gene (CYP1A1) is presumed to be important in pulmonary carcinogenesis and toxicology because its product, the cytochrome P4501A1-dependent (CYP1A1-dependent) monooxygenase, transforms selected xenobiotics (including polycyclic aromatic hydrocarbon procarcinogens in cigarette smoke) to potent carcinogenic metabolites. CYP1A1 messenger RNA (mRNA) expression has not, however, been previously demonstrated in human pulmonary tissue. This report defines CYP1A1 gene expression in normal lung tissue and primary pulmonary carcinoma tissue obtained at thoracotomy from 56 patients with lung cancer. When Northern blot hybridization analyses were performed, 17 of 19 (89%) and zero of five (0%) samples of normal lung tissue from active cigarette smokers and nonsmokers, respectively, expressed the normal 2.8-kilobase CYP1A1 mRNA. In addition, a time-dependent decrease in expression of the CYP1A1 gene was noted in normal lung tissue from individuals who were former smokers, with a decrease in expression occurring as early as 2 weeks following cessation of cigarette smoking. Expression became undetectable in all patients who had stopped smoking more than 6 weeks prior to study. When CYP1A1 gene expression was evaluated in lung cancers, mRNA levels were detectable in one of four (25%) tumors from nonsmokers; two of 24 (8%) tumors from former smokers; and seven of 15 (47%) tumors from cigarette smokers. In addition, an approximately 10-kilobase CYP1A1 RNA species, which was not detectable in normal lung tissue, was observed in five of ten (50%) of the lung cancers that expressed the CYP1A1 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome P-450 CYP1A1↗
PubMed1990

Peripheral lung resistance in normal and asthmatic subjects.

In obstructive lung disease, peripheral airways are a major site of pathologic abnormalities. However, resistance to airflow in small airways in the periphery of the lung accounts for only a small fraction of total airway resistance. Consequently, abnormalities of small airway function may not be readily detected using routine pulmonary function testing. In the present study, resistance of the peripheral lung was examined directly in six normal subjects and nine mildly asthmatic subjects. There were no significant differences between the normal and asthmatic groups in pulmonary function assessed by spirometry (FEV1, FVC) and body plethysmography (specific airway conductance). Direct measurements of peripheral lung function were made using a fiberoptic bronchoscope wedged into a subsegmental, right upper lobe bronchus. Using a double-lumen catheter inserted into the instrument channel of the bronchoscope, pressures (PB) produced by three or more different levels of gas flow (V) (5% CO2 in air) between 50 and 500 ml/min were measured. All pressure measurements were made at a constant lung volume (i.e., functional residual capacity) confirmed by monitoring transpulmonary pressure with an esophageal balloon. The pressure-flow relationship in both normal and asthmatic subjects could be approximated by a straight line through the origin, demonstrating these airways to be relatively nondistensible. Peripheral lung resistance (Rp) was defined by PB/V and averaged for three or more levels of flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Airway Resistance↗
PubMed1990

Evidence for elevated levels of histamine, prostaglandin D2, and other bronchoconstricting prostaglandins in the airways of subjects with mild asthma.

Histamine and certain cyclooxygenase products of arachidonic acid have been implicated as mediators of inflammation and are potent constrictors of human airways. Because asthma may represent manifestations of chronic inflammation of the airways, the levels of histamine and six prostanoid mediators were measured in airway fluids obtained by bronchoalveolar lavage (BAL) of 12 normal, 11 allergic rhinitic, and 15 asymptomatic, allergic asthmatic subjects. Simultaneous profiling of prostanoid mediators in individual samples was performed using gas chromatography-mass spectrometry. Levels of PGD2, 9 alpha,11 beta-PGF2 and PGF2 alpha were 12 to 22 times higher in asthmatic than in normal subjects (p less than 0.01), with concentrations in airway fluids of asthmatic subjects after correction for dilution of 3.8, 0.5, and 1.4 nanomolar, respectively. Levels of PGD2 and 9 alpha,11 beta-PGF2 were increased nearly tenfold in asthmatic subjects compared with those in rhinitic subjects (p less than 0.01), distinguishing the subjects with lower airway disease from those with another atopic condition. Histamine levels were increased fourfold in asthmatic subjects compared with those in normal subjects (p less than 0.001); however, similar increases were found in rhinitic subjects. We conclude that elevated levels of multiple mediators with potent bronchoconstricting activity are present in the airways of subjects with mild asthma, indicating that even mild disease is associated with evidence of airway inflammation. The interactions of bronchoconstricting mediators and airway inflammation may play important roles in the pathogenesis of asthma.

Adult↗
PubMed1990

Histamine acting on a histamine type 1 (H1) receptor increases beta-glucuronidase release from human lung macrophages.

The effects of histamine on lung macrophages have been studied by both biologic and radioligand experiments. After overnight adherence, lung macrophages spontaneously released beta-glucuronidase (beta-G) at a rate of approximately 7 nmol of hydrolyzed substrate/h/million cells. Histamine at low concentrations (10(-9) to 10(-8) M) resulted in a consistent potentiation of this release. The concentration-effect curve of histamine was bell-shaped, reaching an optimum at 10(-9) M, with concentrations greater than 10(-8) M having no significant effect. At a maximally effective concentration (10(-9) M), histamine evoked a 135 +/- 9.6% (mean +/- SE; n = 8, P less than 0.001) potentiation in the total amount of beta-G released during the first 60 min of incubation. This increase in beta-G release represented both a slight increase in beta-G synthesis as well as an increase in the percentage of beta-G released. When the secreted beta-G is expressed as a percentage of total content, histamine (10(-9) M) evoked a 125 +/- 3.2% (mean +/- SE; n = 27, P less than 0.0005) enhancement. The potentiation of beta-G release by histamine was evident after 45 min of incubation and persisted for up to 6 h. The potentiation of beta-G by histamine was sensitive to inhibition by pyrilamine (10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

Tyramine-O-sulfate, in addition to tyrosine-O-sulfate, is produced and secreted by HepG2 human hepatoma cells, but not by 3Y1 rat embryo fibroblasts.

The spent media of HepG2 human hepatoma cells and 3Y1 rat embryo fibroblasts labeled with [35S]sulfate, upon ultrafiltration, were analyzed by a two-dimensional thin-layer separation procedure. Autoradiographs of the cellulose thin-layer plate revealed the presence of tyramine-O-[35S]sulfate in addition to tyrosine-O-[35S]sulfate in spent medium from human hepatoma cells. In contrast, only tyrosine-O-[35S]sulfate was observed in spent medium of 3Y1 rat fibroblasts. Using adenosine, 3'-phosphate, 5'-phospho[35S]sulfate as the sulfate donor, sulfotransferase(s) present in HepG2 cell homogenate catalyzed the sulfation of tyramine to tyramine-O-[35S]sulfate, but not the sulfation of tyrosine to tyrosine-O-[35S]sulfate. Endogenous aromatic amino acid decarboxylase present in HepG2 homogenate was shown to catalyze the decarboxylation of [3H]tyrosine to form [3H]tyramine while attempts to use it for the decarboxylation of tyrosine-O-sulfate to form tyramine-O-sulfate were unsuccessful. These results suggest that tyramine-O-sulfate may be derived from the de novo sulfation of tyramine, instead of the decarboxylation of tyrosine-O-sulfate.

Animals↗
PubMed1989

Bone marrow involvement in non-Hodgkin's lymphoma.

The frequency, pattern, extent and clinical features of bone marrow involvement in 94 cases of non-Hodgkin's lymphoma, classified according to the International Working Formulation, were reviewed. In 30% of cases there was bone marrow involvement by lymphoma at the time of diagnosis. Marrow involvement was most frequently found in the follicular small cleaved cell (57%), diffuse mixed small and large cell (56%), and lymphoblastic (56%) lymphomas. A paratrabecular pattern of marrow involvement tended to occur in low or intermediate grade lymphomas. High grade lymphomas tended to show an interstitial or diffuse pattern of marrow involvement. The results of marrow aspiration and biopsy were of equal value and complementary to each other. In 43% of cases with marrow lymphoma there was peripheral blood involvement at the time of diagnosis. Blood involvement was most frequently found in the lymphoblastic lymphoma (70%). In general, the degree of blood involvement was related to the extent of marrow involvement. Patients with marrow involvement often presented with "B" symptoms and hepatomegaly. Abnormalities in at least one of the blood counts were quite common (93%). A normal blood count made marrow involvement unlikely but not impossible. Other common laboratory findings included hyperimmunoglobulinemia (47%) and elevated serum levels of lactate dehydrogenase (56%). Patients with marrow involvement showed a significantly higher frequency of hepatomegaly and abnormal blood counts, as compared to those with negative marrows (p less than 0.05 and less than 0.001, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1989

Intestinal capillariasis: report of a case.

Infection with Capillaria philippinensis has not been reported in Taiwan before. It is characterized by chronic diarrhea, abdominal pain and muscle wasting. Because the infection results in a severe disease with a high mortality, early diagnosis is very important. A 58-year-old housewife from Ar-Lien village, Kao-Hsiung County, was admitted to the National Taiwan University Hospital in July 1988, after suffering from diarrhea, lower leg edema and weight loss for one year. The initial symptom was epigastric pain followed by watery diarrhea. Thereafter borborygmus, frequent loose stool passage and weakness persisted. The cause of her malabsorption syndrome went undetected until the ova, larvae and adult worms of C. philippinensis were detected in the direct smear of the patient's stool in August 1988. She received mebendazole 200mg b.i.d. for 20 days. Capillaria ova were no longer detected following the third day of medication. At the second month follow-up, her body weight returned to her pre-morbid state and all laboratory findings returned to normal with the exception of mild anemia. The infection source was not clear.

Animals↗
PubMed1988

Spectroscopic characterization of a high-potential monohaem cytochrome from Wolinella succinogenes, a nitrate-respiring organism. Redox and spin equilibria studies.

When purified, a high-potential c-type monohaem cytochrome from the nitrate-respiring organism, Wollinella succinogenes (VPI 10659), displayed a minimum molecular mass of 8.2 kDa and 0.9 mol iron and 0.95 mol haem groups/mol protein. Visible light spectroscopy suggested the presence of an equilibrium between two ligand arrangements around the haem, i.e. an absorption band at 695 nm characteristic of haem-methionine coordination (low-spin form) coexisting with a high-spin form revealed by a band at 619 nm and a shoulder at 498 nm. The mid-point redox potential measured by visible redox titration of the low-spin form was approximately +100 mV. Binding cyanide (Ka = 5 x 10(5) M-1) resulted in the displacement of the methionyl axial residue, and full conversion to a low-spin, cyanide-bound form. Structural features were studied by 300-MHz 1H-NMR spectroscopy. In the oxidized state, the pH dependence of the haem methyl resonances (pH range 5-10) and the magnetic susceptibility measurements (using an NMR method) were consistent with the visible light spectroscopic data for the presence of a high-spin/low-spin equilibrium with a transition pKa of 7.3. The spin equilibrium was fast on the NMR time scale. The haem methyl resonances presented large downfield chemical shifts. An unusually broad methyl resonance at around 35 ppm (pH = 7.5, 25 degrees C) was extremely temperature-dependent [delta(323 K) - delta(273 K) = 7.2 ppm] and was assigned to the S-CH3 group of the axial methionine. In the ferrous state only a low-spin form is present. The haem meso protons, the methyl group and the methylene protons from the axial methionine were identified in the reduced form. The resonances from the aromatic residues (three tyrosines and one phenylalanine) were also assigned. Detailed monitoring of the NMR-redox pattern of the monohaem cytochrome from the fully reduced up to the fully oxidized state revealed that the rate of the intermolecular electronic exchange process was approximately 6 x 10(6) M-1 s-1 at 303 K and pH = 6.31. A dihaem cytochrome also present in the crude cell extract and purified to a homogeneous state, exhibited a molecular mass of 11 kDa and contained 2.43 mol iron and 1.89 mol haem c moieties/mol cytochrome. The absorption spectrum in the visible region exhibited no band at 695 nm, suggesting that methione is not a ligand for either of the two haems. Recovery of only small amounts of this protein prevented more detailed structural analyzes.

Bacteroidaceae↗