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J D Reid

Publications and source records attributed to J D Reid.

At least 19 recordsLinked to original sources
PubMed2002

Current understanding of the function of magnesium chelatase.

Despite the global significance of chlorophylls and other modified tetrapyrroles, many aspects of their biosynthetic pathways are poorly understood. A key enzyme at the branch point between the haem and chlorophyll pathways, magnesium chelatase, couples the free energy of ATP hydrolysis to the insertion of magnesium into porphyrin, a process that is likely to be mediated through protein conformational changes. Conclusions from recent structural and functional studies of individual subunits are combined to provide a mechanistic outline of the full magnesium chelatase complex. Gathering further information presents a considerable challenge, and recent steps towards this goal will be introduced.

Adenosine Triphosphate↗
PubMed2001

Characterization of the binding of deuteroporphyrin IX to the magnesium chelatase H subunit and spectroscopic properties of the complex.

Magnesium protoporphyrin chelatase catalyzes the insertion of a Mg(2+) ion into protoporphyrin IX, which can be considered as the first committed step of (bacterio)chlorophyll synthesis. In the present work, the Mg chelatase H subunits from both Synechocystis and Rhodobacter sphaeroides were studied because of the differing requirements of these organisms for modified cyclic tetrapyrroles. Deuteroporphyrin was shown to be a substrate for Mg chelatase. Analytical HPLC gel filtration was used to show that an H-deuteroporphyrin complex can be reconstituted by incubating the magnesium chelatase H subunit with a molar excess of deuteroporphyrin and that these complexes are monomers. The binding process occurs in the absence of Mg(2+) or ATP or the I or D subunits of Mg chelatase. The emission from Trp residues in the H subunit is partly quenched when deuteroporphyrin is bound. Quantitative analysis of Trp fluorescence quenching led to determination of the K(d) values for deuteroporphyrin binding to BchH from Rb. sphaeroides and ChlH from Synechocystis, which are 1.22 +/- 0.42 microM and 0.53 +/- 0.12 microM for ChlH and BchH, respectively. In the case of ChlH, but not BchH, the K(d) increased 4-fold in the presence of MgATP(2-). Red shifts in absorbance and excitation peaks were observed in the B band of the bound porphyrin in comparison with deuteroporphyrin in solution, as well as reduced yield and red shifts of up to 8 nm in fluorescence emission. These alterations are consistent with a slightly deformed nonplanar conformation of the bound porphyrin. Mg deuteroporphyrin, the product of the Mg chelation reaction, was shown to form a complex with either ChlH or BchH; in each case the K(d) for Mg deuteroporphyrin is similar to that for deuteroporphyrin. The implications of the H-Mg protoporphyrin interaction for the next enzyme in the chlorophyll biosynthetic pathway, Mg protoporphyrin methyltransferase, are discussed.

Bacterial Proteins↗
PubMed2001

Variation in aspects of cysteine proteinase catalytic mechanism deduced by spectroscopic observation of dithioester intermediates, kinetic analysis and molecular dynamics simulations.

The possibility of a slow post-acylation conformational change during catalysis by cysteine proteinases was investigated by using a new chromogenic substrate, N-acetyl-Phe-Gly methyl thionoester, four natural variants (papain, caricain, actinidin and ficin), and stopped-flow spectral analysis to monitor the pre-steady state formation of the dithioacylenzyme intermediates and their steady state hydrolysis. The predicted reversibility of acylation was demonstrated kinetically for actinidin and ficin, but not for papain or caricain. This difference between actinidin and papain was investigated by modelling using QUANTA and CHARMM. The weaker binding of hydrophobic substrates, including the new thionoester, by actinidin than by papain may not be due to the well-known difference in their S2-subsites, whereby that of actinidin in the free enzyme is shorter due to the presence of Met211. Molecular dynamics simulation suggests that during substrate binding the sidechain of Met211 moves to allow full access of a Phe sidechain to the S2-subsite. The highly anionic surface of actinidin may contribute to the specificity difference between papain and actinidin. During subsequent molecular dynamics simulations the P1 product, methanol, diffuses rapidly (over<8 ps) out of papain and caricain but 'lingers' around the active centre of actinidin. Uniquely in actinidin, an Asp142-Lys145 salt bridge allows formation of a cavity which appears to constrain diffusion of the methanol away from the catalytic site. The cavity then undergoes large scale movements (over 4.8 A) in a highly correlated manner, thus controlling the motions of the methanol molecule. The changes in this cavity that release the methanol might be those deduced kinetically.

Catalysis↗
PubMed2000

Modification of cysteine residues in the ChlI and ChlH subunits of magnesium chelatase results in enzyme inactivation.

The enzyme magnesium protoporphyrin chelatase catalyses the insertion of magnesium into protoporphyrin, the first committed step in chlorophyll biosynthesis. Magnesium chelatase from the cyanobacterium Synechocystis PCC6803 has been reconstituted in a highly active state as a result of purifying the constituent proteins from strains of Escherichia coli that overproduce the ChlH, ChlI and ChlD subunits. These individual subunits were analysed for their sensitivity to N-ethylmaleimide (NEM), in order to assess the roles that cysteine residues play in the partial reactions that comprise the catalytic cycle of Mg(2+) chelatase, such as the ATPase activity of ChlI, and the formation of ChlI-ChlD-MgATP and ChlH-protoporphyrin complexes. It was shown that NEM binds to ChlI and inhibits the ATPase activity of this subunit, and that prior incubation with MgATP affords protection against inhibition. Quantitative analysis of the effects of NEM binding on ChlI-catalysed ATPase activity showed that three out of four thiols per ChlI molecule are available to react with NEM, but only one cysteine residue per ChlI subunit is essential for ATPase activity. In contrast, the cysteines in ChlD are not essential for Mg(2+) chelatase activity, and the formation of the ChlI-ChlD-ATP complex can proceed with NEM-treated ChlI. Neither the ATPase activity of ChlI nor NEM-modifiable cysteines are therefore required to form the ChlI-ChlD-MgATP complex. However, this complex cannot catalyse magnesium chelation in the presence of the ChlH subunit, protoporphyrin and Mg(2+) ions. The simplest explanation for this is that in an intact Mg(2+) chelatase complex the ATPase activity of ChlI drives the chelation process. NEM binds to ChlH and inhibits the chelation reaction, and this effect can be partially alleviated by pre-incubating ChlH with magnesium and ATP. We conclude that cysteine residues play an important role in the chelation reaction, in respect of the ChlI-MgATP association, ATP hydrolysis and in the interaction of ChlH with MgATP and protoporphyrin IX.

Adenosine Triphosphatases↗
PubMed1999

Intraoperative intraarterial urokinase in early postoperative stroke following carotid endarterectomy: a useful adjunct.

The purpose of this study was to determine the efficacy of intraoperative intraarterial urokinase (UK) in patients who suffered an acute stroke immediately following carotid endarterectomy (CEA). From January 1995 to March 1998, 823 carotid endarterectomies were performed. The subsequent results showed that intraarterial UK in the setting of early post-CEA neurologic events appears to be safe and may be a useful adjunct to re-exploration in improving neurologic outcomes.

Aged↗
PubMed1998

Identification of a cell type-specific enhancer in the distal 5'-region of the platelet-derived growth factor A-chain gene.

Transient transfection analysis of DNA subfragments from the distal 5'-flanking region of the human platelet-derived growth factor A-chain gene (-18.3 to -1.8 kilobase pairs (kb)) revealed enhancer and silencer elements that contribute significantly to transcriptional regulation. Two adjacent regions (-8.2 to -7.5 kb and -7.5 to -7.0 kb) enhanced transcription of both A-chain and heterologous thymidine kinase promoters, whereas repression was observed in two other nearby regions (-9.9 to -8.2 kb and -7.0 to -5. 9 kb). The -7.5 to -7.0-kb fragment, or J, was the strongest enhancer, and its activity was localized to a 66-base pair element (A-chain cell type-specific enhancer (ACE 66)). ACE66 activity was highly cell type-specific, with greatest activity seen in choriocarcinoma cell lines (4-10-fold enhancement). Progressive 5'- and 3'-deletions of the ACE66 revealed distribution of activity across the element, with nucleotides 1-33 being critical for function. Electrophoretic mobility shift assays revealed cell type-specific patterns of high affinity protein binding to the element. Ethylation interference footprinting of JEG-3 extract localized guanine contacts on nucleotides 1-18 of both strands of the ACE element, whereas more extensive contacts were made with the phosphate backbone (nucleotides 1-32). The ACE66 element is a potent transcriptional regulator in placental cells and represents a valuable model of long distance regulation in a growth factor gene.

Base Sequence↗
PubMed1998

Physiological characterization of the yeast plasma membrane outward rectifying K+ channel, DUK1 (TOK1), in situ.

The major voltage-dependent ion channel in the plasma membrane of Saccharomyces cerevisiae, a conspicuous outwardly rectifying K+ channel, was first dubbed YPK1 and later renamed according to its registered gene names (DUK1, TOK1). It has proven novel in both structure and function. Whole-cell patch-clamp studies of the channel directly on yeast protoplasts now extend our earlier description obtained from isolated patches of yeast membrane (Bertl & Slayman, 1992; Bertl et al., 1993), and provide new data both on the contributions of channel properties to yeast physiology and on possible contributions of molecular structure of channel properties. Three recording tactics produce completely equivalent results and thereby allow great flexibility in the design of experiments: whole-cell voltage clamp with sustained voltage steps (approximately 2.5 sec), whole-cell voltage clamp with slow voltage ramps (5 sec, -40 to +100 mV), and time-averaging of single-channel currents. Activation of Duk1 channels under steady-state conditions is dependent upon ATP in the cytoplasmic solution, and the absence of ATP results in channel "rundown"--decreasing numbers of activable channels--over periods of 10 min to 1 hr from the start of patch recording. Several putative serine- and threonine-phosphorylation sites, as well as a variant ATP-binding fold, exist in the molecule as potential mediators of the ATP effects. The channel runs down similarly following cytoplasmic acidification, but is almost completely insensitive to extracellular pH changes (8.0 to 5.5 tested). This remarkable asymmetry may depend on the protein's strongly asymmetric distribution of histidine residues, with 10 out of 12 predicted to lie close to the membrane-cytoplasm interface. Further data confirm the well-recognized observation that changes of K+ concentration, intracellular or extracellular, can shift the gating voltage of Duk1p in the direction of EK. Among the other alkali-metal cations tested, extracellular Rb+ and Cs(+)--but not Na(+)--substitute almost completely for K+. Extracellular TEA+ inhibits whole-cell K+ currents through Duk1p with a KI of 2.8 mM, and does so probably by reducing the single-channel current.

Cations, Divalent↗
PubMed1998

An aggressive local approach to vascular graft infection.

PURPOSE: To present the use of sartorius myoplasty (SM) and superficial femoral vein (SFV) in a graft-sparing approach to vascular graft infection. METHODS: Twenty-five patients were treated for Szilagyi grade III groin infections during the last 10 years. Fifteen presented early (E), mean 2 months; 10 late (L), mean 6.5 years. Bacteria E/L: Staphylococcus epidermidis 1/7, S aureus 6/0, other gram positive 1/0, gram negative 4/1, mixed 6/0 (one pseudomonas). There were 13 aortofemoral (AF), 5 crossover, and 7 femoral distal reconstructions. SM was used to cover exposed grafts after radical debridement. When the graft was free-floating or bleeding, this segment was removed and replaced with SFV. The remaining infection was controlled with antibiotics. RESULTS: One of 10 patients treated by SM alone required SFV replacement for bleeding. Four of 9 AFs treated by partial SFV replacement +/- SM had persistent infection treated by complete graft removal in 3. Six grafts were removed electively in lower risk situations. There was no perioperative mortality, and no early or late limb loss. CONCLUSIONS: This experience supports an attempt at aggressive local treatment in frail patients.

Aged↗
PubMed1998

A dominant-negative mutant of the platelet-derived growth factor A-chain increases survival of hamsters implanted intracerebrally with the highly invasive CxT24-neo3 glioblastoma cell.

Evidence is accumulating to suggest a role for PDGF in stimulating malignant growth in astrocytoma, although it has been obtained using model systems (growth in 2-dimensional cell culture, athymic nude mice) that do not assess the complex interactions of these tumors with normal brain tissue. In the current study, the highly invasive hamster glioblastoma cell line CxT24-neo3 was used as a model to study the role of platelet-derived growth factor (PDGF) in mediating malignant growth both in vitro and in vivo when implanted directly into the right lateral ventricle of the brain. Co-expression of PDGF B-chain mRNA and PDGF alpha-receptors was detected in these cells, indicating potential for autocrine activation of their growth. CxT24-neo3 cells transfected with wild-type and receptor binding-deficient forms of the PDGF A- and B-chains displayed alterations in their abilities to grow as three-dimensional spheroids, with overexpression of wild-type B-chain resulting in increased spheroid formation, but a decreased rate of spheroid growth. Influence of these PDGF polypeptides on tumor invasion and survival time in vivo was evaluated following implantation of these spheroids in the brain. While all hamsters implanted with control spheroids died within 21 d (average 17 d), those implanted with cells expressing the receptor binding-deficient A-chain survived for much greater periods of time (average 80 d). Modest increases in survival were also seen in cells stably expressing wild-type A-chain (25 d) and mutant B-chain (26 d) proteins. The present study suggests an important role of PDGF in mediating the malignant growth of the CxT24-neo3 cell line in cerebral cortex, possibly via paracrine interactions with normal cortical cell types (i.e., glia, neurons).

Animals↗
PubMed1998

Sarcoidosis in coroner's autopsies: a critical evaluation of diagnosis and prevalence from Cuyahoga County, Ohio.

BACKGROUND AND AIM OF THE WORK: During a histologic study of sarcoidosis in tissues made available from the Cuyahoga County Coroner's autopsy files, the prevalence of this disorder seemed much greater than generally reported. Aims were to evaluate this impression and if confirmed, to explore underlying reasons, including reliability of diagnoses and possible overestimates as well as possible associations with particular population subgroups. METHODS: Cases were retrieved by computer search of diagnoses coded over the past 7 years. Extent of organ involvement was documented and relevant microscope slides reviewed, with consideration of diagnostic criteria generally proposed. Clinical information, occupational risk and evidence for illicit drug use were evaluated. Acceptable cases were categorized by gender, race and age. Crude and standardized prevalences were calculated and related to 1990 US Census figures for the County. RESULTS: On review of 9324 adult autopsies performed for medico-legal reasons, 31 were judged to have sarcoidosis. Standardized prevalence was 320 +/- 72/10(5). Among population subgroups statistically significant differences were not established. However, crude prevalences suggested that females, blacks and younger persons were more likely to have sarcoidosis than males, whites and older subjects, with ratios of 1.6 to 1; 4.7 to 1; and 2.5 to 1, respectively. Overall prevalence was some 10 times greater than indicated from State-wide death certificates. CONCLUSIONS: Sarcoidosis as found in cases autopsied at the/Coroner's Office, Cuyahoga County, is much more prevalent than generally reported. It is an under-appreciated cause of sudden unexpected death. Young black females appear to be most affected, and worthy of further investigation.

Adult↗
PubMed1997

A classical enzyme active center motif lacks catalytic competence until modulated electrostatically.

The cysteine proteinase superfamily is a source of natural structural variants of value in the investigation of mechanism. It has long been considered axiomatic that catalytic competence of these enzymes mirrors the generation of the ubiquitous catalytic site imidazolium-thiolate ion pair. We here report definitive evidence from kinetic studies supported by electrostatic potential calculations, however, that at least for some of these enzymes the ion pair state which provides the nucleophilic and acid-base chemistry is essentially fully developed at low pH where the enzymes are inactive. Catalytic competence requires an additional protonic dissociation with a common pKa value close to 4 possibly from the Glu50 cluster to control ion pair geometry. The pH dependence of the second-order rate constant (k) for the reactions of the catalytic site thiol groups with 4,4'-dipyrimidyl disulfide is shown to provide the pKa values for the formation and deprotonation of the (Cys)-S-/(His)-Im+H ion pair state. Analogous study of the reactions with 2,2'-dipyridyl disulfide reveals other kinetically influential ionizations, and all of these pKa values are compared with those observed in the pH dependence of kcat/Km for the catalyzed hydrolysis of N-acetylphenylalanylglycine 4-nitroanilide. The discrepancy between the pKa value for ion pair formation and the common pKa value close to 4 related to generation of catalytic activity is particularly marked for ficin (pKa 2.49 +/- 0.02) and caricain (pKa 2.88 +/- 0.02) but exists also for papain (pKa 3.32 +/- 0.01).

Binding Sites↗
PubMed1996

Site-directed mutagenesis of the N-linked glycosylation site in platelet-derived growth factor B-chain results in diminished intracellular retention.

Pulse-chase analysis of human platelet-derived growth factor (PDGF) B-chain was conducted in stably transfected Chinese hamster ovary cells to determine precisely the kinetics of processing, intracellular trafficking and secretion. Newly synthesized 31 kDa monomers of the B-chain (p31) dimerized rapidly via disulfide bonds to a p54 species (t1/2 < 30 min). The p54 dimer was processed to a group of intracellular, cell surface (suramin-releasable) and secreted forms whose rates of appearance and disappearance from the cell were measured over a 48 h period. The newly synthesized p31 species was quantitatively converted to p27 by treatment with endoglycosidase H, consistent with efficient N-glycosylation at a site in the N-terminal propeptide region (Asn63-Met64-Thr65). Interruption of B-chain glycosylation by oligodeoxynucleotide-directed mutagenesis resulted in a significant increase in suramin-releasable forms at the cell surface (p34-38) and a concomitant decrease in accumulation of an intracellular p24 species. The glycosylation-defective mutant exhibited slight increases in receptor binding and mitogenic activity. Our results suggest that N-linked glycosylation of the B-chain is not important for formation of mitogenically active protein, but that it plays a role in early intracellular sorting and proteolytic processing events.

Animals↗
PubMed1996

PWP2, a member of the WD-repeat family of proteins, is an essential Saccharomyces cerevisiae gene involved in cell separation.

WD-repeat proteins contain four to eight copies of a conserved motif that usually ends with a tryptophan-aspartate (WD) dipeptide. The Saccharomyces cerevisiae PWP2 gene, identified by sequencing of chromosome III, is predicted to contain eight so-called WD-repeats, flanked by nonhomologous extensions. This gene is expressed as a 3.2-kb mRNA in all cell types and encodes a protein of 104 kDa. The PWP2 gene is essential for growth because spores carrying the pwp2 delta 1::HIS3 disruption germinate before arresting growth with one or two large buds. The growth defect of pwp2 delta 1::HIS3 cells was rescued by expression of PWP2 or epitope-tagged HA-PWP2 using the galactose-inducible GALI promoter. In the absence of galactose, depletion of Pwp2p resulted in multibudded cells with defects in bud site selection, cytokinesis, and hydrolysis of the septal junction between mother and daughter cells. In cell fractionation studies, HA-Pwp2p was localized in the particulate component of cell lysates, from which it would be solubulized by high salt and alkaline buffer but not by nonionic detergents or urea. Indirect immunofluorescence microscopy indicated that HA-Pwp2p was clustered at multiple points in the cytoplasm. These results suggest that Pwp2p exists in a proteinaceous complex, possibly associated with the cytoskeleton, where it functions in control of cell growth and separation.

Base Sequence↗
PubMed1996

Changes in voltage activation, Cs+ sensitivity, and ion permeability in H5 mutants of the plant K+ channel KAT1.

KAT1 is a voltage-dependent inward rectifying K+ channel cloned from the higher plant Arabidopsis thaliana [Anderson, J. A., Huprikar, S. S., Kochian, L. V., Lucas, W. J. & Gaber, R. F. (1992) Proc. Natl. Acad. Sci. USA 89, 3736-3740]. It is related to the Shaker superfamily of K+ channels characterized by six transmembrane spanning domains (S1-S6) and a putative pore-forming region between S5 and S6 (H5). The 115 region between Pro-247 and Pro-271 in KAT1 contains 14 additional amino acids when compared with Shaker [Aldrich, R. W. (1993) Nature (London) 362, 107-108]. We studied various point mutations introduced into H5 to determine whether voltage-dependent plant and animal K+ channels share similar pore structures. Through heterologous expression in Xenopus oocytes and voltage-clamp analysis combined with phenotypic analysis involving a potassium transport-defective Saccharomyces cerevisiae strain, we investigated the selectivity filter of the mutants and their susceptibility toward inhibition by cesium and calcium ions. With respect to electrophysiological properties, KAT1 mutants segregated into three groups: (i) wild-type-like channels, (ii) channels modified in selectivity and Cs+ or Ca2+ sensitivity, and (iii) a group that was additionally affected in its voltage dependence. Despite the additional 14 amino acids in H5, this motif in KAT1 is also involved in the formation of the ion-conducting pore because amino acid substitutions at Leu-251, Thr-256, Thr-259, and Thr-260 resulted in functional channels with modified ionic selectivity and inhibition. Creation of Ca2+ sensitivity and an increased susceptibility to Cs+ block through mutations within the narrow pore might indicate that both blockers move deeply into the channel. Furthermore, mutations close to the rim of the pore affecting the half-activation potential (U1/2) indicate that amino acids within the pore either interact with the voltage sensor or ion permeation feeds back on gating.

Amino Acid Sequence↗