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S K Chapman

Publications and source records attributed to S K Chapman.

At least 91 records · Page 5Linked to original sources
PubMed1989

The role of the C-terminal tail of flavocytochrome b2.

A flavocytochrome b2 (L-lactate dehydrogenase) mutant was constructed in which the C-terminal tail (23 amino acid residues) had been deleted (Gly-489----Stop). This tail appears to form many intersubunit contacts in the tetrameric wild-type protein, and it was expected that its removal might lead to the formation of monomeric flavocytochrome b2. The isolated tail-deleted mutant enzyme (TD-b2), however, was found to be tetrameric (Mr 220,000). TD-b2 shows Km and kcat. values (at 25 degrees C and pH 7.5) of 0.96 +/- 0.06 mM and 165 +/- 6 s-1 respectively compared with 0.49 +/- 0.04 mM and 200 +/- 10 s-1 for the wild-type enzyme. The kinetic isotope effect with [2-2H]lactate as substrate seen for TD-b2, with ferricyanide as electron acceptor, was essentially the same as that observed for the wild-type enzyme. TD-b2 exhibited loss of activity during turnover in a biphasic process. The rate of the faster of the two phases was dependent on L-lactate concentration and at saturating concentrations showed a first-order deactivation rate constant, kf(deact.), of 0.029 s-1 (at 25 degrees C and pH 7.5). The slower phase, however, was independent of L-lactate concentration and gave a first-order deactivation rate constant, ks(deact.), of 0.01 s-1 (at 25 degrees C and pH 7.5). This slower phase was found to correlate with dissociation of FMN, which is one of the prosthetic groups of the enzyme. Thus fully deactivated TD-b2, which was also tetrameric, was found to be completely devoid of FMN. Much of the original activity of TD-b2 could be recovered by re-incorporation of FMN. Thus the C-terminal tail of flavocytochrome b2 appears to be required for the structural integrity of the enzyme around the flavin active site even though the two are well separated in space.

DNA Mutational Analysis↗
PubMed1989

Structural basis for the kinetic differences between flavocytochromes b2 from the yeasts Hansenula anomala and Saccharomyces cerevisiae.

To understand the structural basis for the different catalytic behaviour of the flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala we have cloned and sequenced the gene encoding the latter. We have compared the amino acid sequences of the mature proteins in the context of the known crystal structure of S. cerevisiae flavocytochrome b2. Overall there is 60% sequence identity, but two surface loops in particular are strikingly different in primary structure and net charge.

Amino Acid Sequence↗
PubMed1989

High-level expression of fully active yeast flavocytochrome b2 in Escherichia coli.

Wild-type flavocytochrome b2 (L-lactate dehydrogenase) from the yeast Saccharomyces cerevisiae and three singly substituted mutant forms (F254, R349 and K376) have been expressed in the bacterium Escherichia coli. The enzyme expressed in E. coli contains the protohaem IX and flavin mononucleotide (FMN) prosthetic groups found in the enzyme isolated from yeast, has an electronic absorption spectrum identical with that of the yeast protein and an identical Mr value of 57,500 estimated by SDS/polyacrylamide-gel electrophoresis. N-Terminal amino-acid-sequence data indicate that the flavocytochrome b2 isolated from E. coli begins at position 6 (methionine) when compared with mature flavocytochrome b2 from yeast. The absence of the first five amino acid residues appears to have no effect on the enzyme-catalysed oxidation of L-lactate, since Km values for the yeast- and E. coli-expressed wild-type enzymes were identical within experimental error. The F254 mutant enzyme expressed in E. coli also showed kinetic parameters essentially the same as those found for the enzyme from yeast. The R349 and K376 mutant enzymes had no activity when expressed in either yeast or E. coli. The yield of flavocytochrome b2 from E. coli is estimated to be between 500- and 1000-fold more than from a similar wet weight of yeast (this high level of expression results in E. coli cells which are pink in colour). The increased yield has allowed us to verify the presence of FMN in the R349 mutant enzyme. The advantages of E. coli as an expression system for flavocytochrome b2 are discussed.

Escherichia coli↗
PubMed1989

Effect of acyclovir on radiation- and chemotherapy-induced mouth lesions.

Several chemotherapeutic regimens and radiation therapy, if delivered to the oral mucosa, are associated with a high frequency of mouth lesions. The cause of this side effect is not known for certain, but in past studies it has sometimes been associated with the ability to culture herpes simplex virus type 1 from the mouth. In a double-blind prospective trial, patients with head and neck tumors treated with chemotherapy or radiation therapy were treated with either acyclovir or placebo. Although the frequency of culture-positive herpes simplex virus was low in the untreated group, it was significantly lower, zero, in the acyclovir-treated group. However, there were no differences in the frequency or type of mouth lesions experienced by patients receiving either radiation or chemotherapy who were taking acyclovir or placebo. These results suggest that herpes simplex virus is not a frequent cause or complication of oral lesions afflicting this patient population.

Acyclovir↗
PubMed1988

Probing the active site of flavocytochrome b2 by site-directed mutagenesis.

The three-dimensional structure of flavocytochrome b2 (L-lactate dehydrogenase) from bakers' yeast (Saccharomyces cerevisiae) has recently been solved at 0.24-nm resolution [Mathews & Xia (1987) in Flavins and flavoproteins, Walter de Gruyter, Berlin, pp. 123-131]. We have used this structural information to investigate the roles of particular amino acid residues likely to be involved in the oxidation of L-lactate by kinetic analysis of mutant enzymes generated by site-directed mutagenesis of the isolated gene. The hydroxyl group of Tyr254 was expected to be important for the abstraction of the hydroxyl proton of L-lactate in the oxidation to pyruvate. Replacement of this tyrosine by phenylalanine reduced kcat from 190 +/- 3 s-1 (25 degrees C, pH 7.5) to 4.3 +/- 0.1 s-1. This substitution had, however, no discernable effect on Km for lactate (0.54 +/- 0.03 mM for the mutant compared with 0.49 +/- 0.03 mM for the wild-type enzyme). Arg376 was expected to be essential for productive binding and orientation of L-lactate. Replacing Arg376 with lysine abolished all detectable activity. A total loss of enzymic activity was also observed when Lys349, thought likely to stabilize the anionic form of the flavin hydroquinone, was replaced by arginine. An amino acid residue replacement at a distance from the active site, Ala306 to serine, had a minor but significant effect on kcat (reduced from 190 s-1 to 160 s-1) and Km (increased from 0.49 mM to 0.83 mM) presumably arising from small conformational effects. The implications of these results are discussed in relation to the mechanism of L-lactate oxidation.

Binding Sites↗
PubMed1986

Preferred sites on cytochrome c for electron transfer with two positively charged blue copper proteins, Anabaena variabilis plastocyanin and stellacyanin.

Rate constants for the reactions of horse cytochrome c (E'0 of +260 mV) with the copper proteins Anabaena variabilis plastocyanin (E'0 of +360 mV) used as oxidant and stellacyanin (E'0 of +187 mV) used as reductant have been determined at 25 degrees C, pH 7.5 and 7.0, respectively, and an ionic strength of 0.10 M (NaCl). These rate constants were also measured with eight different singly substituted 4-carboxy-2,6-dinitrophenyl (CDNP) horse cytochrome c derivatives, modified at lysine-7, -13, -25, -27, -60, -72, -86, or -87 and with the trinitrophenyl (TNP) derivative modified at lysine-13. The influence of the modifications on the bimolecular rate constants for these reactions defines the region on the protein that is involved in the electron-exchange reactions and demonstrates that the preferred site is at or near the solvent-accessible edge of the heme prosthetic group on the "front" surface of the molecule. Both reactions are strongly influenced by the lysine-72 modification to the left of the exposed heme edge and, to this extent, behave similar to the earlier studied reaction with azurin. These effects span only an order of magnitude in rate constants and are thus many times smaller than those for the physiological protein redox partners of cytochrome c. While the preferred sites of reaction on the surface of cytochrome c for small inorganic complexes appear to be dependent only on the net charge of the reactants, with the copper proteins additional factors intervene. These influences are discussed in terms of hydrophobic patches and the distribution of charges on the surface of the four copper proteins so far examined.

Binding Sites↗
PubMed1983

Preferred sites for electron transfer between cytochrome c and iron and cobalt complexes.

The kinetics of oxidation of eight different singly substituted 4-carboxy-2,6-dinitrophenyl (CDNP) horse ferrocytochromes c, modified at lysine 7, 13, 25, 27, 60, 72, 86, or 87, and of one trinitrophenyl horse ferrocytochrome c, modified at lysine 13, by the 3- and 3+ inorganic complexes hexacyanoferrate(III) (Fe(CN)6(3-) ) and tris(1,10-phenanthroline)cobalt(III) (Co(phen)3(3+) ) have been characterized. The influence of the modified residues on the bimolecular rate constants for these reactions define the protein molecular surface involved. The site of electron exchange for both oxidants appears to be the solvent accessible edge of the heme prosthetic group or a closely related structure on the "front" surface of the molecule. The reaction with Fe(CN)6(3-) is most strongly influenced by modification of lysine 72, a residue to the left of the exposed heme edge. (CDNP lysine 72 cytochrome c yields a 3.6-fold decrease in the bimolecular rate constant, as compared to that for the native protein.) However, it is the region around lysine 27, to the right of the heme edge, that is most influential in the reaction with Co(phen)3(3+). (CDNP-lysine 27 cytochrome c exhibits a 7.3-fold increase in the rate constant, as compared to that for the native protein.) The kinetics of reaction of the CDNP-lysine 13, 60, 72, and 87 modified cytochromes c with Fe(CN)5(4-aminopyridine)2- as oxidant and Fe(CN)5(4-aminopyridine)3- and Fe(CN)5-(imidazole)3- as reductants have also been determined and further illustrate the influence of electrostatics on the kinetics of such protein-small molecule electron exchanges.

Chemical Phenomena↗
PubMed1983

Interaction of cytochrome c with the blue copper proteins, plastocyanin and azurin.

Bimolecular rate constants have been determined for the reactions of native horse cytochrome c, eight 4-carboxy-2,6-dinitrophenyl (CDNP-) cytochromes c singly modified at lysines 7, 13, 25, 27, 60, 72, 86, or 87 and one 2,3,6-trinitrophenyl cytochrome c singly modified at lysine 13, with the blue copper proteins, plastocyanin (from parsley leaves) and azurin (from Pseudomonas aeruginosa). Plastocyanin, a protein having a negative charge of about -7, yields a bimolecular rate constant with native ferrocytochrome c of 1.5 x 10(6) M-1 S-1, which decreases with the modified cytochromes c to a minimum of 7.5 x 10(5) M-1 S-1 for the CDNP-lysine 13 derivative. Conversely azurin, a protein with an overall negative charge of only about -1 to -2, exhibits bimolecular rate constants with native ferrocytochrome c of 6.6 x 10(3) M-1 S-1 at pH 6.1 and 4.0 x 10(3) M-1 S-1 at pH 8.6, which increase upon modification of the cytochrome c to a maximum of 4.1 x 10(4) M-1 S-1 at pH 6.1 and 2.7 x 10(4) M-1 S-1 at pH 8.6, for the CDNP-cytochrome c modified at lysine 72. This behavior indicates that: 1) the reaction of cytochrome c occurs at a negatively charged site on plastocyanin, whereas azurin behaves as a positively charged reactant, the electrostatics governing to a large extent the relative reactivities of the modified cytochromes c; 2) in both cases the interaction domain on cytochrome c is located on the "front" surface of the protein and encompasses the solvent accessible edge of the heme prosthetic group, as is the case for all the reactions of cytochrome c with its mitochondrial protein redox partners, as well as for small inorganic redox complexes; and 3) the bimolecular rate constants for plastocyanin and azurin are orders of magnitude slower and the effects of lysine modifications far smaller than for the reactions with physiological systems, indicating that: (a) the electric fields generated by the reactants do not align them, prior to electron transfer, as effectively as for the physiological reaction partners of cytochrome c; and (b) there is an absence of a precise molecular fit between cytochrome c and the nonphysiological redox partners.

Azurin↗
PubMed1982

Combined light and electron microscope in routine histopathology.

We report our experience with a prototype combined light and electron microscope (the LEM 2000) with particular reference to its application to routine surgical histopathology. We found its major advantages over conventional transmission electron microscopies were due to the large grid size (7 mm diameter), low magnification capacity (x 250), and the built-in microprocessor for recording areas of interest. These features combine to reduce sampling errors and greatly facilitate orientation and relocation of fields of diagnostic importance.

Histological Techniques↗
PubMed1980

Antiproliferative effects of inhibitors of polyamine synthesis in tumors of neural origin.

The antiproliferative properties of inhibitors of polyamine synthesis were evaluated in cultured neuroblastoma and glioma cells. The diamines (1,3-propanediamine, 1,5-pentanediamine, and 1,6-hexanediamine) dramatically decreased neuroblastoma replication and inhibited the rate-limiting enzyme, ornithine decarboxylase. Glioma cells were less sensitive to the diamines in spite of significant drug-induced decreases in enzyme activity. The fact that ornithine decarboxylase was inhibited in both cell lines with different effects on proliferation suggests that the activity of other enzymes in polyamine biosynthesis may be altered selectively by these inhibitors.

Animals↗
PubMed1978

A search for the function of human carbonic anhydrase B.

Because of the very high activity and abundance of human red cell carbonic anhydrase C (carbamate hydrolase, EC 4.2.1.1), it seemed likely that the second isozyme, B, might not be essential for CO2 metabolism. It was then found that physiological concentrations of Cl- inhibited catalysis of CO2 hydration by the B enzyme (but not by type C), suggesting further that type B does not function in vivo as a carbonic anhydrase. The versatility of the catalytic activity of carbonic anhydrase for a number of 'artificial' substrates suggested that enzyme B may be utilized in reactions of intermediary metabolism. A number of hydration, dehydration, decarboxylation, kinase, and phosphatase systems were tested to determine a possible physiological function for the enzyme. Results with eighteen possible substrates were negative and the possibility is discussed that mammalian carbonic anhydrase B is an evolutionary accident.

Carbonic Anhydrases↗
PubMed1978

Acetazolamide and renal ammoniagenesis.

The effect of acetazolamide on ammonia-producing enzyme systems was determined in vitro at concentrations comparable to those which have been shown to abolish ammonium excretion in vivo. No change in the activity of glutaminase or gamma-glutamyl transpeptidase could be observed at concentrations up to 0.2 mM acetazolamide, and concentrations up to 1 mM were without effect on D-glutamyltransferase activity. Therefore, the effect of acetazolamide to abolish ammonium excretion cannot be explained by an action of the drug to inhibit ammoniagenesis.

Acetazolamide↗
PubMed1977

The identification of particulate matter in biological tissues and fluids.

Sections, 1-2 micron thick, of Araldite embedded tissue were prepared to provide a link between optical and electron microscopy for the identification of crystals. This technique permits examination of individual particles by means of polarising light microscopy, electron microscopy, X-ray energy spectroscopy and electron diffraction. It provides a reliable method for routine and research studies. It has led to the positive identification of individual crystals of calcium pyrophosphate dihydrate and hydroxyapatite in synovial fluids. The method can be applied to the identification of other particulate matter in a variety of biological specimens.

Aged↗
PubMed1975

The effect of variations in objective focal length on electron microscope performance.

Standard specimens were examined in a transmission electron microscope with a top entry stage. Using a range of objective focal lengths, measurements of contrast, resolution and magnification range were made. Focal lengths up to 14 mm were examined. The contrast was found to increase with increasing focal length, being twelve and a half times greater at 14 mm focal length than at the normal high resolution mode of 2 mm focal length. Resolution, determined by the fresnel fringe method decreased from 0-3 nm to 3-5 nm. The photographic image mangnification was reduced from X 1000-X500,000 at 2 mm focal length to X100-X50,000 at 14 mm focal length.

Microscopy, Electron↗