Direct measurement of metal ion chelation in the active site of human ferrochelatase.

The final step in heme biosynthesis, insertion of ferrous iron into protoporphyrin IX, is catalyzed by protoporphyrin IX ferrochelatase (EC 4.99.1.1). We demonstrate that pre-steady state human ferrochelatase (R115L) exhibits a stoichiometric burst of product formation and substrate consumption, consistent with a rate-determining step following metal ion chelation. Detailed analysis shows that chelation requires at least two steps, rapid binding followed by a slower (k approximately 1 s-1) irreversible step, provisionally assigned to metal ion chelation. Comparison with steady state data reveals that the rate-determining step in the overall reaction, conversion of free porphyrin to free metalloporphyrin, occurs after chelation and is most probably product release. We have measured rate constants for significant steps on the enzyme and demonstrate that metal ion chelation, with a rate constant of 0.96 s-1, is approximately 10 times faster than the rate-determining step in the steady state (kcat = 0.1 s-1). The effect of an additional E343D mutation is apparent at multiple stages in the reaction cycle with a 7-fold decrease in kcat and a 3-fold decrease in kchel. This conservative mutation primarily affects events occurring after metal ion chelation. Further evaluation of structure-function data on site-directed mutants will therefore require both steady state and pre-steady state approaches.

Patency of superficial femoral vein employed as a crossover femoral artery bypass conduit.

This study assesses the patency of superficial femoral vein used as a crossover femoral artery bypass conduit in patients presenting either with localized groin sepsis, generalized sepsis or in patients with occluded or heavily diseased superficial femoral artery outflow. Twenty patients were followed prospectively with femoral crossover grafts constructed of superficial femoral vein. Twelve patients presented with sepsis and 8 with chronic ischemia from iliac artery occlusion and severely diseased superficial femoral artery outflow. Graft patency was assessed with regular duplex ultrasound examination. There was one perioperative death. Six patients died during the follow-up period. Mean follow-up time was 24.3 months. No graft occluded or required revision. There was no limb loss, graft infection, or graft hemorrhage. Superficial femoral vein offers an effective femoral crossover bypass graft in patients with either localized/generalized sepsis or disadvantaged outflow tracts.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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).

Functional comparison of plant inward-rectifier channels expressed in yeast.

Functional expression of plant ion channels in the yeast Saccharomyces cerevisiae is readily demonstrated by the successful screening of plant cDNA libraries for complementation of transport defects in especially constructed strains of yeast. The first experiments of this sort identified two potassium-channel genes from Arabidopsis thaliana, designated KAT1 and AKT1 (Anderson et al., 1992; Sentenac et al., 1992), both of which code for proteins resembling the Shaker superfamily of K(+) channels in animal cells. Patch-clamp analysis, directly in yeast, of the two channel proteins (Kat1 and Akt1) reveals both functional similarities and functional differences: similarities in selectivity and in normal gating kinetics; and differences in time-dependent effects of ion replacement, in the affinities of blocking ions, and in dependence of gating kinetics on extracellular K(+). Kat1, previously described in yeast (Bertl et al., 1995), is about 20-fold more permeable to K(+) than to Na(+) or NH(+)(4), shows K(+)-independent gating kinetics, and is blocked with moderate effectiveness (30-50% at 10 mM) by barium and tetraethylammonium (TEA(+)) ions. Akt1, by contrast, is weakly inhibited by TEA(+), more strongly inhibited by Ba(2+), and very strongly inhibited by Cs(+). Furthermore Na(+) and NH(+)(4), while having about the same permeance to Akt1 as to Kat1, have delayed effects on Akt1: brief replacement of extracellular K(+) by Na(+) enhances by nearly 100% the subsequent K(+) currents after sodium removal; and brief replacement of K(+) by NH(+)(4) reduces subsequent K(+) currents by nearly 75%. Furthermore, lowering of extracellular K(+) concentration, by replacement with osmotically equivalent sorbitol, significantly retards the opening of Akt1 channels; that is, the gating kinetics for Akt1 are clearly influenced by the concentration of permeant ions. In this respect, Akt1 resembles the native yeast outward rectifier, Ypk1 (Duk1; Reid et al., 1996). The data suggest that all of the ions tested bind within the open channels, such that the weakly permeant species (Na(+), NH(+)(4)) are easily displaced by K(+), but the blocking species (Cs(+), Ba(2+), TEA(+)) are not easily displaced. With Akt1, furthermore, the permeant ions bind to a modulator site where they persist after removal from the medium, and through which they can alter the channel conductance. Extracellular K(+) itself also binds to a modulator site, thereby enhancing the rate of opening of Akt1.

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.

Prosthetic-vein composite graft with remote popliteal arteriovenous fistula.

BACKGROUND: Inadequate autogenous vein is often the limiting factor in femoral-distal reconstruction. Sequential grafts, distal arteriovenous fistulae (AVF), and autogenous cuffs have been recommended in this setting. This paper describes a new method of composite reconstruction, enhanced with a popliteal AVF. PATIENTS AND METHODS: Data was collected prospectively on 13 patients (14 limbs) who presented with severe ischemia (rest pain or ulcer 6, gangrene 8). The average age was 75 years. Eleven of the reconstructions were to tibial outflow tracts. The novelty of the technique is in the construction of a controlled AVF between the upper end of an autogenous vein graft and the popliteal vein. An uncomplicated autogenous anastomosis is created distally. The synthetic graft is placed between the common femoral artery and the vein graft distal to the AVF. The flow is balanced between the fistula and the outflow tract by narrowing the fistula while monitoring the pressure in the graft system. RESULTS: Patency was assessed by color duplex scan. Two grafts occluded (at 5 and 7 months) and 1 was revised at 12 months. Mean peak systolic velocity in the synthetic portion was 130 cm/s. Limb edema has not been a problem. There were 2 perioperative cardiac deaths. Mean follow up is 15 months (range 6 to 36). CONCLUSION: The addition of a controlled AVF below the synthetic portion of a composite graft appears promising when vein is in limited supply.