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Direct (unmediated) electrochemistry of the photosynthetic "blue" copper protein plastocyanin may be readily achieved at an edge-oriented pyrolytic graphite electrode over the pH range 4-8. Promotion of rapid charge transfer by Mg2+ ions (

The cytochrome b5 tail is a 43-residue membrane-embedded domain that is responsible for anchoring the catalytic domain of cytochrome b5 to the endoplasmic reticulum membrane. Different models for the structure of the membrane domain of cytochrome b5 have been proposed, including a helical hairpin and a single transmembrane helix. In the present study, CD spectroscopy was used to investigate the conformation of the tail in different environments, and as a function of temperature, with the goal of understanding the nature of the membrane-bound conformation. Whereas residue property profiling indicates that bending of a helix in the middle of the peptide might be possible, the experimental results in small unilamellar vesicles and lysophosphatidylcholine micelles are more consistent with a single transmembrane helix. Furthermore, although there is evidence for some refolding of the polypeptide with temperature, this is not consistent with a hairpin-to-transmembrane transition. Rather, it appears to represent an increase in helical content in fluid lipid environments, perhaps involving residues at the ends of the transmembrane segment.

The nuclear magnetic resonance solution structure of alpha-conotoxin SI has been determined at pH 4.2. The 36 lowest energy structures show that alpha-conotoxin SI exists in a single major solution conformation and is stabilized by six hydrogen bonds. Comparisons are made between the SI solution structure and the solution and crystal structures of alpha-conotoxin GI. Surprisingly, a high degree of similarity between the backbone conformations of the GI crystal and the SI solution structures is seen in the region of lowest sequence homology, namely residues Gly-8 to Ser-12. This similarity is more surprising when considering that in SI a proline replaces the Arg-9 found in GI. The correspondence in conformation in this region provides the definitive evidence that it is the loss of the arginine basic charge at residue 9 which determines the differences in toxicity between GI and SI, rather than any changes in conformation induced by the cyclic proline residue.

Determination of 15N-NMR relaxation rates has allowed the characterisation of the dynamic properties of 55 1H-15N bond vectors in a soluble haem-binding domain of bovine microsomal ferricytochrome b5 consisting of the first 104 amino acid residues. Measurements of heteronuclear [1H]-15N-NMR nuclear Overhauser effects, and 15N-NMR longitudinal and transverse relaxation rates have been analysed using both model-free and reduced spectral density mapping approaches, demonstrating the application of these methods to a paramagnetic system. Analysis reveals that, for those regions which have been assessed, the polypeptide backbone of ferricytochrome b5 is largely rigid (average S2 = 0.80), and that minimal internal backbone motion occurs on the sub-nanosecond timescale. In contrast, motions on the microsecond to millisecond timescale, especially pronounced for the loop region extending from residues 28 to 31, and which may be of biological relevance, are indicated.

Chymopapain M, the monothiol cysteine proteinase component of the chymopapain band eluted after chymopapains A and B in cation-exchange chromatography, was isolated from the dried latex of Carica papaya and characterized by kinetic and chromatographic analysis. This late-eluted chymopapain is probably a component of the cysteine proteinase fraction of papaya latex discovered by Schack [(1967) Compt. Rend. Trav. Lab. Carlsberg 36, 67-83], named papaya peptidase B by Lynn [(1979) Biochim. Biophys. Acta 569, 193-201] and partially characterized by Polgár [(1981) Biochim. Biophys. Acta 658, 262-269] and is the enzyme with unusual specificity characteristics (papaya proteinase IV) that Buttle, Kembhavi, Sharp, Shute, Rich and Barrett [Biochem. J. (1989) 261, 469-476] claimed to be a previously undetected cysteine proteinase eluted from a cation-exchange column near to the early-eluted chymopapains. A study of the time-dependent chromatographic consequences of thiol-dependent proteolysis of the components of papaya latex is reported. Chymopapain M was isolated by (i) affinity chromatography followed by separation from papain using cation-exchange f.p.l.c. on a Mono S HR5/5 column and (ii) cation-exchange chromatography followed by an unusual variant of covalent chromatography by thiol-disulphide interchange. The existence in chymopapain M of a nucleophilic interactive Cys/His catalytic-site system analogous to those in papain (EC 3.4.22.2) and other cysteine proteinases was deduced from the characteristics shape of the pH-second-order rate constant (k) profiles for its reactions with 2,2′-dipyridyl disulphide and ethyl 2-pyridyl disulphide. Analysis of the pH-k data for the reactions of chymopapain M with the 2-pyridyl disulphides and with 4,4′-dipyridyl disulphide permits the assignment of molecular pKa values of 3.4 and 8.7 to the formation and subsequent dehydronation of the Cys-S-/His-Im+H state of the catalytic site and reveals three other kinetically influential ionizations with pKa values 3.4, 4.3 and 5.6. The pH-dependences of kcat./Km for the hydrolysis of N-acetyl-L-Phe-Gly-4-nitroanilide at 25.0 degrees C and I0.1 M catalysed by chymopapain M and papain were determined. For both enzymes, little catalytic activity (5-7% of the maximal) develops consequent on formation of the catalytic site Cys-S-/His-Im+H ion-pair state (across pKa 3.4 for both enzymes). For papain, full expression of Kcat./Km for the uncharged substrate requires only the additional hydronic dissociation with pKa 4.2. By contrast, full expression of kcat./Km for chymopapain M requires additional hydronic dissociation with pKa values of 4.3 and 5.6.(ABSTRACT TRUNCATED AT 400 WORDS)

Thermally induced denaturation has been measured for both oxidised and reduced forms of the tryptic fragment of bovine microsomal cytochrome b5 using spectrophotometric methods. In the oxidised state, the tryptic fragment of cytochrome b5 (Ala7-Lys90) denatures in a single cooperative transition with a midpoint temperature (Tm) of approximately 67 degrees C (pH 7.0). The reduced form of the tryptic fragment of cytochrome b5 shows a higher transition temperature of approximately 73 degrees C at pH 7.0 and this is reflected in the values of delta Hm, delta Sm and delta(delta G) of approximately 310kJ.mol-1, 900J.mol-1.K-1 and 5 kJ.mol-1. Increased thermal stability is demonstrated for a variant protein that contains the first 90 amino acid residues of cytochrome b5. These novel increases in stability are observed in both redox states and result from the presence of six additional residues at the amino-terminus. The two forms of cytochrome b5 do not differ significantly in structure with the results suggesting that the reorganisation energy (lambda) of the variant protein, as measured indirectly from redox-linked differences in conformational stability, is small. Consequently the reported subtle differences in reactivity between variants of cytochrome b5 may result from the presence of additional N-terminal residues on the surface of the protein.

1H-NMR spectroscopy has been used to measure the rate of unimolecular electron exchange between cytochrome c molecules in protein aggregates stabilised by the addition of sodium hexametaphosphate. The average intracomplex electron exchange rate is measured from line broadening of hyperfine-shifted resonances of ferricytochrome c in an equimolar mixture of reduced and oxidised protein. The line-broadening due to electron exchange is significantly greater than that due to protein aggregation and reaches a maximum value between 1 – 2 mol hexametaphosphate/mol protein. Significantly the exchange-induced broadening is a first-order process and is directly proportional to the size of the cytochrome c oligomer. From the temperature dependence of exchange broadening the activation enthalpy was estimated to be 75.8 kJ mol−1 whereas the activation entropy was 295 J mol−1 K−1 for a dimer of cytochrome c at a hexametaphosphate/protein molar ratio of 1. Both activation parameters decrease in magnitude as the order of the cytochrome c oligomer increases. The rates of intracomplex electron exchange in Saccharomyces cerevisiae iso-2 and Candida krusei cytochromes c are lower than that of the horse protein, implying that primary sequence plays a fundamental part in determining the rate of exchange. The relevance of these observations is discussed in terms of the function of cytochrome c.

The association of the tryptic fragment of bovine microsomal cytochrome b5 with cytochrome c has been studied by one- and two-dimensional 1H-NMR spectroscopy. The association of cytochromes to form protein complexes is apparent from the increase in linewidths for resonances of ferricytochrome b5 as well as small perturbations in their chemical shifts that occur upon increasing the cytochrome c/b5 molar ratio. The changes in the chemical shifts of hyperfine shifted resonances of ferricytochrome b5 with increasing ratios of ferricytochrome c indicate the formation of binary 1:1 complexes and ternary 1:2 complexes. Similarly, titrations of the linewidth of resolved resonances of ferricytochrome b5 are consistent with stoichiometries of 1:1 and 1:2 for complexes formed between cytochromes b5 and c. Surprisingly, in the 1:1 complex, mobility is shown to be a function of ionic strength. Two-dimensional correlated spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy (NOESY) of the binary complex formed between ferricytochrome b5 and c indicate that the positions of many resonances attributable to amino acids are unaltered by protein association, although distinctive chemical shift changes are detected in the alpha-CH of the haem C17 propionate. The protein complex detected by NMR is discussed with respect to the model for the binary complex proposed by Salemme and possible mechanisms of electron transfer.

There is, as yet, no strong culture of research in primary care and much of the existing research is conceived and undertaken by people outside primary care. The poor implementation of research findings may, in part, be owing to the fact that those delivering the service are not involved in asking or answering questions that are relevant to their practice. This paper reports how three practices constructed a research agenda based on the unanswered questions of their primary care teams. The research questions prioritised by the teams tended towards patient behaviour and service organisation rather than clinical issues. This contrasts with national research priorities. The process has contributed towards the development of a culture of enquiry among team members. Other primary care teams may benefit from a similar approach. National research priority setting in primary care should take more account of the unanswered questions of primary care teams.

A comparison of the primary sequences of the heme binding domains of bovine and rat microsomal cytochrome reveal differences at only six residues. These residues must therefore provide the origin for the observed variation in the ratio of the heme orientational isomers, the equilibrium constant of which ranges from ∼9 in the bovine protein to ∼1.6 for rat cytochrome b 5 . Residues 7, 20, 21, and 30 are distant from the exposed heme edge whilst Leu 27 and Phe 78 are located close to different parts of the porphyrin macrocycle. 1 H NMR spectra of the heme and heme ligand resonances of a recombinant tobacco cytochrome b 5 extending from Gly 1 to Lys 89 suggest, in combination with NMR data acquired for other forms of cytochrome b 5 and an inspection of their sequence homology, that the identity of residue 78 influences the relative ratios of heme isomers. The Gly 1 -Lys 89 domain of tobacco cytochrome b 5 has two equally abundant heme orientational isomers but retains the leucine side chain at position 27 whilst phenylalanine 78 is replaced by tyrosine. A more direct role for residue 78 in modulating the heme ratio is shown by site directed mutagenesis of bovine microsomal cytochrome b 5 where the mutation Phe 78 >Tyr shifts the equilibrium constant for the heme orientational isomers from 9 to 3.5. Whilst the ratio is clearly shifted towards that exhibited by the rat protein the incomplete transition suggested the involvement of other residues. The mutation of Leu 27 >Val was shown to result in a slightly smaller change in ratios of each isomer (from 9 to 4.0). Together these results point to the importance of these residues in modulating the ratio of heme isomers.

One-dimensional and two-dimensional 1H-NMR methods and paramagnetic difference spectroscopy have defined cation binding domains on the surface of the tryptic fragment of microsomal cytochrome b5. The addition of tris(ethylenediamine) chromium(III) [Cr(en)3(3+)] to solutions of ferricytochrome b5 reveals at least three distinct sites on the surface of the protein to which highly charged cations may bind (20 mM phosphate pH 7.0, T = 300 K). Surprisingly only one of these sites is located close to the haem edge region of the protein, whilst the remaining two sites are more remote. Site I contains the exposed haem C13 propionate and a series of carboxylate residues that includes glutamates 37, 38, 43, 44, and 48. Sites II and III are located away from the haem edge region and are delineated by the broadening of aromatic resonances of histidines 26 and 80. Further investigation of the interaction between Cr(en)3(3+) and cytochrome b5 using two-dimensional double-quantum-filtered correlated spectroscopy shows that resonances assigned to Glu59, Asp60, Glu79, Asp82 and Asp83 are broadened with the distribution of these charged side chains correlating with the relaxation broadening observed from one-dimensional experiments. In a binary complex with ferricytochrome c, Cr(en3(3+) broadens many cytochrome b45 resonances including the haem propionates, His26, Ala54, Thr55 and His80. Although the pattern of line-broadening of resonances at sites II and III is unaltered by complex formation, cytochrome c shields residues at site I, the haem edge site. The results indicate that the interaction between cytochrome b5 and c in a binary complex involves multiple protein configurations.

An internal water molecule (designated WAT166) is found in iso-1-cytochrome c which is part of a redox-state-dependent hydrogen bond network. The position of this water molecule with respect to the polypeptide fold can be altered or even displaced by site-directed mutagenesis leading to structural perturbations and associated changes in redox potential. Using saturation transfer 1H-NMR methods, this study measures changes in the electron transfer reactivity for three variants of yeast iso-1-cytochromes c in which the position of this water molecule is altered. In particular, the reverse electron transfer rate is measured within a complex formed between either wild-type or variant yeast iso-1-cytochromes c and the tryptic fragment of bovine liver microsomal cytochrome b5. For three variants of yeast iso-1-cytochrome c the rate constants measured by saturation transfer are wild-type (Asn52, E0 = 270 mV, kex = 0.3 s-1), Asn52----Ala (E0 = 240 mV, kex = 0.6 s-1), Asn52----Ile (E0 = 220 mV, kex = 1.0 s-1). The first-order rates are compared with that of a fourth variant Phe82----Gly which has been measured previously (E0 = 220 mV, kex = 0.7 s-1). An analysis of the variation in the observed cross exchange rate using Marcus theory shows that these changes can be predicted quantitatively by the shift in redox potential that accompanies mutagenesis. So, although the perturbation of the internal water molecule by mutagenesis alters both the structure and redox potential of cytochrome c, surprisingly it does not significantly influence the intrinsic electron transfer reactivity of the protein. Studies of the activation parameters suggests that a variation of temperature changes both delta G* and also the prefactor. These data are discussed in terms of models involving dynamic molecular recognition between proteins.

In the presence of the highly charged hexametaphosphate anion, horse heart cytochrome c aggregates to form stable protein complexes. The formation of protein aggregates has been detected by high-resolution 1H-NMR spectroscopy from an increase in the linewidth of resolved ferricytochrome c resonances with hexametaphosphate concentration. Alternatively, analytical ultracentrifugation reveals protein association from the increase in apparent sedimentation coefficients of cytochrome c in the presence of equimolar hexametaphosphate. Protein aggregation is dependent on the concentration of background electrolyte since in the range 10–150 mM sodium cacodylate alternative stabilisation of dimeric and trimeric complexes was observed by both NMR and analytical ultracentrifugation. A model is proposed for the mechanism of protein aggregation caused by polyphosphate binding to the surface of cytochrome c.

Das Denken und Handeln des Strassburger Reformators Martin Bucer (1491-1551) ist uberaus vielfaltig und komplex. Die vorliegende Studie bietet erstmalig eine koharente Gesamtdarstellung dieser Gedankenwelt und ihrer praktischen Umsetzung. Der Terminus regnum Christi, ein Schlusselbegriff der bucerschen Theologie, steht im Zentrum der Arbeit, wobei insbesondere das Verhaltnis zwischen Reich Christi und Obrigkeit analysiert wird. Anhand der Themenkomplexe Bilder, Bildung, Messe, Dissidenten, Zucht und Interim werden die jeweiligen Positionen Bucers und des Strassburger Rats, ihre Zusammenarbeit wie auch ihre Kontroversen aufgezeigt. Im Rahmen der Themenstellung beschaftigt sich die Studie auch mit Bucers Wirken ausserhalb von Strassburg. Analysiert werden das reformatorische Engagement in den freien Reichsstadten Ulm und Augsburg sowie im Erzbistum Koeln, die Beratertatigkeit zugunsten des Landgrafen Philipp von Hessen (u. a. in der Frage nach der Tolerierung der Juden) sowie die Bemuhungen um die sichtbare Einheit des regnum Christi in der Zeit der Religionsgesprache.

The lipid content of a Photosystem II preparation, derived by Triton X-100 fractionation of spinach thylakoid membranes, which retained high rates of oxygen evolution was examined. It was found that the detergent treatment resulted in a preferential loss of digalactosyldiacylglycerol. The effect on this preparation of exogenously added lipids, or lipid mixtures, purified from thylakoids was investigated. Addition of total polar lipid extract stimulated the rates of oxygen evolution, the same effect being achieved with isolated digalactosyldiacylglycerol or phosphatidylcholine alone. The stimulation of oxygen evolution was dependent on the degree of unsaturation of the lipids used and on the relative amounts of acidic lipids present in the mixtures. Addition of isolated acidic lipids alone resulted in a complete inhibition of oxygen evolution. The results are discussed in terms of the possible involvement of thylakoid lipids in the molecular organisation of the Photosystem II oxygen-evolving system.

The site-specific chemical modification of horse heart cytochrome c at Lys-13 and -72 using 4-chloro-3,5dinitrobenzoic acid (CDNB) increases the electron self-exchange rate of the protein. In the presence of 0.24 M cacodylate (pH* 7.0) the electron self-exchange rate constants, k,,, measured by a ‘H NMR saturation transfer method at 300 K, are 600,6 x lo3 and 6 x 10“ M-‘.s-’ for native, CDNP-K13 and CDNP-K72 cytochromes c respectively. Repulsive electrostatic interactions, which inhibit cytochrome c electron selfexchange, are differentially affected by modification. Measurements of ‘H NMR line broadening observed with partially oxidised samples of native cytochrome c show that ATP and the redox inert multivalent anion Co(CN)z- catalyse electron self-exchange. At saturation a limiting value of - 1.4 x lo5 M-i.s-’ is observed for both anions. Cytochrome c Chemical modiJication Electron transfer NMR Protein-protein interaction ATP

Two dimensional 1 H NMR spectroscopy is used to examine the structure and mobility of cytochrome b 5 in solution. The assignment of many residues and the interpretation of nuclear Overhauser effects (NOEs) in both redox states allow definition of secondary structural elements. Comparison with X-ray diffraction data shows that differences between crystal and solution structures are small. The dynamics of the protein are examined and the protein is shown to be more mobile than cytochrome c . The relationship of the structure and dynamics to the electron transfer function of cytochrome b 5 is discussed.

There exists wide variation in practice regarding the use of chaperones for physical examination of the patient in primary care. Guidelines agree that a chaperone should be offered or used whenever a patient is undergoing an intimate examination. This paper aims to gauge patient views on the use of chaperones in primary care. Focus groups were used to identify themes surrounding the use of chaperones in primary care. Questionnaires were then mailed to 404 women and 400 men aged 16 years to 65 years identified from the lists of three research practices in the Northern and Yorkshire Region. The most important factor for the patient in an intimate examination is the attitude of the doctor; the patient views the offer of a chaperone as a sign of respect by the doctor. The development of shared decision-making within a consultation involving an intimate examination should be seen as more important than a rigid approach towards using a chaperone.