Biomedical subjects

C Ho

Publications and source records attributed to C Ho.

At least 109 records · Page 6Linked to original sources

PubMed1995

Stopped-flow kinetic and biophysical studies of membrane-associated D-lactate dehydrogenase of Escherichia coli.

The enzyme kinetics of the FAD-containing membrane-associated D-lactate dehydrogenase (D-LDH) of Escherichia coli have been investigated by stopped-flow spectroscopy. The reduction of D-LDH by the substrate, D-lactate, exhibits a two-stage behavior as observed by the absorbance change for the enzyme-bound FAD. The fast stage with a maximum rate of 400 s-1 represents the rapid formation of the enzyme-substrate complex and the formation of the equilibrium between the oxidized and the reduced enzyme-substrate complexes. The slow stage, which occurs on the order of 0.36 s-1, represents the slow release of the product, pyruvate, from the reduced enzyme. The formation of a D-LDH semiquinone radical was not observed during the oxidation of D-lactate by D-LDH at 25 degrees C. However, during the subsequent electron transfer from the reduced enzyme to a nitroxide spin-label, a one-electron acceptor, an enzyme intermediate has been observed and identified by both optical and EPR spectroscopies as an anionic semiquinone. Results from 1H-NMR spectroscopic studies suggest the possible formation of a substrate carbanion when D-lactate is bound at the active site of D-LDH.

2,6-Dichloroindophenol↗

PubMed1995

Nature and environment of the sulfhydryls of membrane-associated D-lactate dehydrogenase of Escherichia coli.

Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid), has been used to titrate D-lactate dehydrogenase (D-LDH), a respiratory flavoenzyme of Escherichia coli. All six of the possible sulfhydryls titrate in the presence of 2% sodium dodecylsulfate, showing that D-lactate dehydrogenase does not contain any -S-S- bridges. In the native state, only two sulfhydryls are accessible in buffer and only one in the presence of lipid. Single-site mutations of each of the six cysteines of D-lactate dehydrogenase have been prepared. Each of the purified mutant proteins has full activity, demonstrating that an -SH group is not essential to the FAD-driven redox reaction. Ellman's titrations of the mutant proteins have led to the identification of cysteines 65, 146, 156, and 256 in the amino-terminal end as those containing the sulfhydryls that are not accessible in buffer or in buffer plus lipid. The cysteine at 422 is titrated only partially in buffer, while in buffer containing lipid, a necessary factor for full enzymatic activity, its sulfhydryl is inaccessible to the reagent. Cysteine 492 has been identified as containing the sulfhydryl that is accessible to the reagent under both conditions.

Bacterial Outer Membrane Proteins↗

PubMed1995

Expression of a functionally active human renal sodium-calcium exchanger lacking a signal sequence.

The Na+-Ca2+ exchanger is an unusual membrane transport protein as it contains an NH2-terminal signal sequence which is co-translationally removed in the endoplasmic reticulum during synthesis. To determine if the signal sequence was essential for biosynthesis, mutations were introduced in the NH2 terminus of the cDNA coding for the human renal Na+-Ca2+ exchanger in order to alter processing of the protein. To prevent cleavage of the signal sequence during biosynthesis, the last residue of the consensus signal sequence, Ala-1, was changed to Phe. Deletion mutants were also constructed to encode for exchangers which lacked the signal sequence, the signal sequence and the first extracellular loop, or all of the NH2 terminus including the first transmembrane segment of the mature protein. These mutants were expressed in HEK 293 cells and assayed for Na+-Ca2+ exchange activity. Mutants lacking either a signal sequence or containing a noncleavable signal sequence were still targeted to the plasma membrane, where they exhibited Na+-Ca2+ exchange activity. By contrast, the mutants which had more than the signal sequence deleted did not demonstrate any exchange activity. These mutants were, however, still integrated into the membrane and were resistant to alkali extraction. These results show that the signal sequence is not essential for biogenesis of the Na+-Ca2+ exchanger and suggests that the molecule contains one or more internal signal sequences for insertion into the membrane during biosynthesis.

Amino Acid Sequence↗

PubMed1995

A novel low oxygen affinity recombinant hemoglobin (alpha96val--> Trp): switching quaternary structure without changing the ligation state.

Using our Escherichia coli expression plasmid (pHE2) in which synthetic human alpha and beta-globin genes are coexpressed with the E. coli methionine aminopeptidase gene under the control of separate tac promoters, we have constructed a new artificial hemoglobin in which the valine residue at position 96 of the alpha chain, located in the alpha 1 beta 2 subunit interface, has been replaced by a tryptophan residue using site-directed mutagenesis. We have determined the oxygen-binding properties of this recombinant hemoglobin, r Hb (alpha 96Val-->Trp), and have used proton nuclear magnetic resonance spectroscopy to investigate its tertiary structure around the heme group and the quaternary structure in the alpha 1 beta 2 subunit interface. This artificial hemoglobin shows a low oxygen affinity, but high cooperativity in oxygen binding, and exhibits no unusual subunit dissociation when ligated. Molecular dynamics simulations suggest that the unique oxygen-binding property of r Hb (alpha 96Val-->Trp) may be due to an extra hydrogen bond between alpha 96Trp and beta 99Asp in the alpha 1 beta 2 subunit interface in the deoxy form. Despite the replacement of a small amino acid residue, valine, by a large tryptophan residue in the alpha 1 beta 2 subunit interface, this artificial hemoglobin shows very similar tertiary structure around the heme pockets and quaternary structure in the alpha 1 beta 2 subunit interface compared to those of human normal adult hemoglobin. Another unique feature of this artificial hemoglobin is that the ligated form, e.g. carbonmonoxy form, of this hemoglobin in the oxy-quaternary structure can be converted to the deoxy-like quaternary structure by the addition of an allosteric effector, inositol hexaphosphate, as well as by lowering the temperature in the absence of inositol hexaphosphate, without changing its ligation state. Thus, this recombinant hemoglobin can be used to gain new insights regarding the nature of subunit interactions in the alpha 1 beta 2 interface and the molecular basis for the allosteric mechanism of hemoglobin.

Base Sequence↗

PubMed1995

Hydration and order in lipid bilayers.

The relationship between membrane lipid bilayer hydration and acyl chain order was investigated using time-resolved fluorescence spectroscopy. The degree of hydration in the head group region was assessed from fluorescence lifetime data along with fluorescence intensity measurements in D2O, relative to H2O buffer, using N-(5-dimethylaminonaphthalene-1-sulfonyl)dipalmitoylphosphatidylethan ola mine (dansyl-PE). The degree of hydration in the acyl chain region was estimated from its effect on the fluorescence lifetime of 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl] carbonyl]-3-sn-phosphatidylcholine (DPH-PC), and acyl chain order was determined from time-resolved anisotropy measurements of the DPH-PC. Comparisons of sn-2 unsaturation with sn-1,2 diunsaturation in phosphatidylcholine (PC) bilayers with the same number of double bonds/PC revealed a marked difference in interchain hydration and acyl chain order but little difference in terms of head group hydration. For diunsaturated dioleoyl-PC (DOPC) bilayers with two double bonds/PC, the DPH-PC fluorescence lifetime data indicated a greater level of interchain hydration than 1-palmitoyl-2-docosahexaenoyl-PC (PDPC) with six double bonds/sn-2 chain. By contrast, the head group hydration for DOPC was markedly less than for PDPC. A similar lack of correlation of effects on the two regions of the bilayer was found with cholesterol, it having opposite effects on interchain and head group hydration. When DPH-PC fluorescence lifetime data for bilayers composed of a range of different lipids was plotted as a function of acyl chain order, a strong correlation of interchain hydration with acyl chain order was revealed that was independent of lipid composition.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrolysis↗

PubMed1995

Direct activation of protein kinase C by 1 alpha,25-dihydroxyvitamin D3.

The key metabolite of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 (1,25-D3), induces rapid cellular responses that constitute a so-called "non-genomic" response. This effect is distinguished from its "classic" genomic role in calcium homeostasis involving the nuclear 1,25-D3 receptor. Evidence is presented that protein kinase C (PKC) is directly activated by 1,25-D3 at physiological concentrations (EC50 = 16 +/- 1 nM). The effect was demonstrable with single PKC-alpha, -gamma, and -epsilon isoform preparations, assayed in a system containing only purified enzyme, substrate, co-factors, and lipid vesicles, from which it is inferred that a direct interaction with the enzyme is involved. The finding that calcium-independent isoform PKC-epsilon was also activated by 1,25-D3 shows that the calcium binding C2 domain is not required. The level of 1,25-D3-induced activation, paired with either diacylglycerol or 4 beta-12-O-tetradecanoylphorbol-13-acetate, was greater than that achievable by any individual activator alone, each at a saturating concentration, a result that implies two distinct activator sites on the PKC molecule. Phosphatidylethanolamine present in the lipid vesicles potentiated 4 beta-12-O-tetradecanoylphorbol-13-acetate- and diacylglycerol-induced PKC activities, whereas 1,25-D3-induced activity decreased, consistent with 1,25-D3-activated PKC possessing a distinct conformation. The results suggest that PKC is a "membrane-bound receptor" for 1,25-D3 and that it could be important in the control of non-genomic cellular responses to the hormone.

Animals↗

PubMed1995

In vivo dynamic MRI tracking of rat T-cells labeled with superparamagnetic iron-oxide particles.

Dynamic MRI tracking of rat T-cells in vivo is performed in rat testicles after labeling isolated rat T-cells in vitro with superparamagnetic dextran-coated iron-oxide particles, BMS180549. Tissue inflammation induced by the local injection of the calcium ionophore, A23187, is used to attract labeled T-cells. Gradient-echo MR images of rat testicles show a statistically significant decrease (4%) of the signal intensity in areas of injection of A23187 as early as 30 min after intravenous infusion of 2 x 10(8) labeled T-cells. The signal change reaches its maximum (6-7% decrease) at about 60-120 min after cell infusion. T2-mapping also shows a decrease of T2 in the areas with A23187. Image quantitation, which includes a chemical-shift effect, significantly enhances the sensitivity for detection of superparamagnetically labeled T-cells. Localization of labeled T-cells in rat testicles has been verified by fluorescence microscopy studies of T-cells co-labeled with a lipophilic fluorescent carbocyanine dye, 1,1-dioctadecyl-3,3,3',3'-tetramethyl-lindocarbocyanine perchlorate. These results represent the first successful demonstration of dynamic tracking of specific cells in vivo using MRI.

Animals↗

PubMed1995

1H-NMR investigation of the oxygenation of hemoglobin in intact human red blood cells.

Using improved selective excitation methods for protein nuclear magnetic resonance (NMR), we have conducted measurements of the oxygenation of hemoglobin inside intact human red blood cells. The selective excitation methods use pulse shape-insensitive suppression of the water signal, while producing uniform phase excitation in the region of interest and, thus, are suitable for a wide variety of applications in vivo. We have measured the areas of 1H-NMR resonances of the hyperfine-shifted, exchangeable N delta H protons of the proximal histidine residues of the alpha- and beta-chains in deoxyhemoglobin (63 and 76 ppm downfield from the proton resonance of 2,2-dimethyl-2-silapentane-5-sulfonate (DSS), respectively), which are sensitive to the paramagnetic state of the iron, and for which the alpha- and beta-chain resonances are resolved, and from the ring current-shifted gamma 2-CH3 protons of the distal valine residues in oxyhemoglobin (2.4 ppm upfield from DSS), which are sensitive to the conformation of the heme pocket in the oxy state. We have found that the proximal histidine resonances are directly correlated with the degree of oxygenation of hemoglobin, whereas the distal valine resonances appear to be correlated with the conformation in the heme pocket that occurs after the binding of oxygen, in both the presence and absence of 2,3-diphosphoglycerate. In addition, from the proximal histidine resonances, we have observed a preference for the binding of oxygen to the alpha-chain (up to about 10%) of hemoglobin over the beta-chain in both the presence and absence of 2,3-diphosphoglycerate. These new results obtained in intact erythrocytes are consistent with our previous 1H-NMR studies on purified human normal adult hemoglobin. A unique feature of our 1H-NMR method is the ability to monitor the binding of oxygen specifically to the alpha- and beta-chains of hemoglobin both in solution and in intact red blood cells. This information is essential to our understanding of the molecular basis for the hemoglobin molecule serving as the oxygen carrier in vertebrates.

Carboxyhemoglobin↗

PubMed1995

A mouse model of human familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism.

Mice lacking the calcium-sensing receptor (Casr) were created to examine the receptor's role in calcium homeostasis and to elucidate the mechanism by which inherited human Casr gene defects cause diseases. Casr+/- mice, analogous to humans with familial hypocalciuric hypercalcemia, had benign and modest elevations of serum calcium, magnesium and parathyroid hormone levels as well as hypocalciuria. In contrast, Casr-/- mice, like humans with neonatal severe hyperparathyroidism, had markedly elevated serum calcium and parathyroid hormone levels, parathyroid hyperplasia, bone abnormalities, retarded growth and premature death. Our findings suggest that Casr mutations cause these human disorders by reducing the number of functional receptor molecules on the cell surface.

Animals↗

PubMed1995

Severe controlled cortical impact in rats: assessment of cerebral edema, blood flow, and contusion volume.

Controlled cortical impact (CCI) is a contemporary model of experimental cerebral contusion. We examined the cerebrovascular and neuropathologic effects of a severe CCI in rats. The utility of magnetic resonance imaging (MRI) for the assessment of contusion volume after severe CCI was also established. Severe CCI (3.0 mm depth, 4 m/sec velocity) to the left (L) parietal cortex was produced in anesthetized (isoflurane/N2O/O2), intubated, and mechanically ventilated male Sprague-Dawley rats (n = 58). Physiologic parameters were controlled. The time course of alterations in edema [L-R% brain water (% BW) in 3-mm coronal sections through injured and contralateral hemispheres, wet-dry weight] was evaluated at 2 h, 24 h, 48 h, and 7 days posttrauma. Local cerebral blood flow (ICBF, measured in 8 structures in each hemisphere by autoradiography) was evaluated at 2 h, 24 h, and 7 days. Contusion volume (measured by histology and image analysis) was assessed at 14 days and measured in 6 rats by both MRI and histology. The survival rate after severe CCI was 96.2%. The L-R difference in % BW increased to 1.69 +/- 0.18% at 2 h, 3.00 +/- 0.08% at 24 h, 2.69 +/- 0.09% at 48 h, and 0.94 +/- 0.21% at 7 days. These values all differed from the control (p < 0.05). The % BW was greater at 24 h and 48 h than at 2 h and 7 days (p < 0.05). Marked reductions in ICBF were limited to structures in the injured hemisphere and were observed in the parietal cortex (2 and 24 h), subcortical white matter (2 and 24 h), and hippocampus (2 h), (p < 0.05) vs control rats. In the contusion core, ICBF was 19.4 +/- 8.8 mL 100 g-1 min-1 at 24 h (p = 0.011 vs normal). Necrosis was seen in large portions of the parietal cortex and subcortical white matter, and portions of the hippocampus and thalamus. Contusion volume was 47.8 +/- 9.2 mm3, which represented 14.4 +/- 2.1% of the traumatized hemisphere. Estimates of contusion volume by MRI and histology were closely correlated (r = 0.941, p < 0.017). Severe CCI in rats is accompanied by contusion, reproducible edema, and marked hypoperfusion, involving over 14% of the injured hemisphere, and can be produced with minimal mortality. T2-weighted MRI successfully and noninvasively identifies contusion volume in this model.

Animals↗

PubMed1995

A novel P2-purinoceptor expressed by a subpopulation of astrocytes from the dorsal spinal cord of the rat.

1. Astrocytes from the dorsal spinal cord express P2-purinoceptors which, when stimulated, produce a rise in the intracellular level of free Ca2+ ([Ca2+]i). Previously we have found that the P2Y class of receptor is expressed by nearly all astrocytes from the dorsal horn. To determine whether other metabotropic P2-purinoceptor classes are also present, in this study we investigated the effects of UTP. 2. Application of UTP (1-500 microM, 5-20 s) produced a transient rise in [Ca2+]i in a subpopulation of astrocytes. The magnitude of the peak increase in [Ca2+]i was dependent upon UTP concentration and the EC50 was found to be 5.2 +/- 0.2 microM. Ca2+ responses were maximum at 100 microM UTP. 3. The rise in [Ca2+]i in response to UTP was not affected by removal of extracellular Ca2+. On the other hand, application of the sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase inhibitor, thapsigargin, abolished responses to UTP. These findings indicate that UTP stimulates the release of Ca2+ from a thapsigargin-sensitive intracellular pool. 4. The Ca2+ response to UTP was unaffected by treatment with pertussis toxin, suggesting that UTP responses may be mediated via a pertussis toxin-insensitive G protein. 5. While all cells tested (n = 52) responded to the P2Y-purinoceptor agonist, 2-methylthio-ATP, only a subpopulation of astrocytes (n = 67/93) was responsive to UTP. The presence of UTP-sensitive and UTP-insensitive cells requires the existence of two discrete types of receptor. One receptor, expressed by UTP-insensitive cells, appears to be activated selectively by 2-methylthio-ATP. 6. To investigate whether UTP and 2-methylthio-ATP activate a common type of receptor in UTP-responsive cells, a cross-desensitization strategy was used. Desensitization with prolonged exposure to a high concentration of 2-methylthio-ATP failed to affect responses to UTP and vice versa, indicating that receptors activated by UTP are distinct from those activated by 2-methylthio-ATP. 7. The P2-purinoceptor antagonist, suramin (100 microM), blocked Ca2+ responses to UTP and to 2-methylthio-ATP. 8. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), has been reported to block responses mediated by P2X- and P2Y-purinoceptors in other systems and therefore we investigated its effects on responses to 2-methylthio-ATP and to UTP. PPADS was found to block Ca2+ responses to 2-methylthio-ATP in a concentration-dependent manner with an IC50 of 0.92 +/- 0.1 microM. PPADS also blocked UTP-evoked responses and the IC50 was 7.2 +/- 1.9 microM. At a concentration of 10 microM, PPADS produced a rightward shift in the dose-response curve for UTP and did not affect the maximum response. 9. Calcium responses evoked by the muscarinic agonist, carbachol, were unaffected either by suramin (100 microM) or by PPADS (50 microM). 10. The present results indicate the presence of a novel class of metabotropic P2U-purinoceptor in dorsal spinal astrocytes. In contrast to P2Y-purinoceptors, the P2U-purinoceptor is expressed only by a subpopulation of astrocytes and its sensitivity to suramin and PPADS distinguish this receptor from P2U-purinoceptors found in other tissues.

Adenosine Triphosphate↗

PubMed1995

Laser resurfacing in pigmented skin.

BACKGROUND: Laser skin resurfacing is a very promising new treatment for solar elastosis and acne scars. The Ultrapulse carbon dioxide (CO2) laser and the SilkTouch flashscanner allow precise depth control and char-free ablation. Earlier studies have been done mainly on Caucasian skin types I and II, but very little data were available on pigmented skin. OBJECTIVE: The purpose of this study is to assess the efficacy and safety of laser skin resurfacing in skin types III and IV. METHODS: Thirty Asian and Hispanic patients were treated for facial rhytides and acne scars. All patients were instructed to use tretinoin cream 0.05%, hydroquinone 5%, and desonide 0.1% cream nightly for 2-4 weeks prior to the laser treatment. The Ultrapulse 5000C CO2 laser with the Truespot 3-mm collimated handpiece at the setting of 250-450 mJ per pulse, or the Silk-Touch flashscanner at the setting of 5-7 W, 0.2-second pulse duration, and 4-mm (M) spot size, was used. Patients were evaluated clinically, and global response was assessed by both clinicians and patients. RESULTS: The facial rhytides and acne scars improved 25-50% in all patients after one laser treatment. The most common side effects were persistent erythema that resolved on the average of 6 weeks. Hyperpigmentation occurred, but was reduced with regular use of tretinoin, hydroquinone, and desonide cream both pre- and postoperatively along with use of broad spectrum sunscreen after treatments. CONCLUSION: Laser skin resurfacing can be used to treat facial rhytides and acne scars in skin phototypes III and IV. When proper pre- and postoperative management is implemented, the risk of dyspigmentation can be reduced.

Acne Vulgaris↗

PubMed1995

Mini-slit graft hair transplantation using the Ultrapulse carbon dioxide laser handpiece.

BACKGROUND: The new Ultrapulse carbon dioxide (CO2) laser technology has added a new dimension to many cosmetic surgery procedures including hair transplantation. Early reports by Unger and David (Laser Hair Transplantation. J Dermatol Surg Oncol 1994;20:515-21) have been encouraging with the potential of minimal bleeding, ease of placing transplanted grafts, and an overall shortened operative time. A 2-mm slit handpiece has been recently created to expedite this procedure. OBJECTIVE: The purpose of this study is to further investigate the use and efficiency of the new Ultrapulse CO2 laser slit handpiece in hair transplants. METHOD: Mini-slit graft hair transplants using the new Ultrapulse CO2 laser slit handpiece were done on 25 patients in 30 transplant sessions. Donor minigrafts were obtained by the strip harvesting technique using a triple-blade scalpel. Approximately 200-400 recipient slits were made with the 2-mm slit handpiece at the laser setting of 350 mJ, 12 W, 0.8 seconds per pulse. RESULTS: All grafts were easily placed into recipient sites with minimal bleeding and charring. The procedure was done in half the time of the conventional non-laser technique. Postoperatively, patients were quite satisfied with little pain and swelling. Histologic exams of the laser-treated slits showed minimal adjacent tissue necrosis. Long-term follow-up visits showed good regrowth of hair in these grafts. CONCLUSION: The new Ultrapulse CO2 laser slit handpiece proved to be an effective tool for mini-slit graft hair transplantation.

Adult↗

PubMed1995

Routine urinalysis in renal transplant patients.

The performance of two commercially available urine dipstick tests was evaluated to exclude significant bacteriuria in midstream urine samples from patients attending a renal transplant outpatient clinic. The use of dipsticks to reduce the necessity for routine microscopy and culture was also assessed. Consecutive urine samples (n = 497) from 121 patients were examined for leucocytes, nitrite, and blood with Multistix* 10SG (Ames) and Nephur test (Boehring) dipsticks. All urine samples also underwent routine microscopy and culture. The sensitivities of the Nephur test and Multistix were 83% and 82%, respectively; the specificities were 28% and 40%, respectively; the negative predictive values were 85% and 88%, respectively; and the positive predictive values were 24% and 29%, respectively. Underlying conditions and treatments in renal patients may have led to relatively low sensitivity and positive predictive values. Traditional microscopy and culture methods are more reliable for renal transplant recipients.

Adult↗

PubMed1995

Removal of carcinoma cells from contaminated bone marrow using the lipophilic cation rhodamine 123.

Autologous bone marrow transplants for solid tumor treatment are severely limited by the potential presence of residual cancer cells in the reinfused bone marrow and can lead to future tumor recurrence. This article presents a novel method of removing carcinoma cells from bone marrow with contaminating cancer cells. This method is based on our previous studies demonstrating that carcinoma cells have a higher uptake of lipophilic cations such as rhodamine 123 than their normal epithelial counterparts. When the relative differences in rhodamine 123 uptake are quantified, carcinoma cell lines demonstrated a 7.4-21 times greater uptake of rhodamine 123 than normal mouse bone marrow cells. More important, when normal bone marrow cells and carcinoma cell lines are mixed to simulate carcinoma-contaminated bone marrow, individual cell populations continue to exhibit characteristic and identifiable relative differences (10-20 times) in rhodamine 123 uptake. Differential sorting of bone marrow/carcinoma cell mixtures with respect to rhodamine 123 fluorescence intensity resulted in the removal of 95-99% of the "contaminating carcinoma cells." The recovered bone marrow cells were fully viable as ascertained by their ability to form splenic colonies. In our preliminary experiments, sorted bone marrow cells transplanted into lethally irradiated C57BL6 mice allowed the mice to survive for more than 8 months. In light of these promising results, we propose that lipophilic cations may play a role in the purification of autologous bone marrow used in transplants for patients with advanced solid tumors.

Animals↗

PubMed1994

Restoring allosterism with compensatory mutations in hemoglobin.

Abnormal human hemoglobins (HBs) with amino acid substitutions in the alpha 1 beta 2 interface have very high oxygen affinity and greatly reduced cooperativity in O2 binding compared to normal human Hb. In such abnormal Hbs with mutations at position beta 99, the intersubunit hydrogen bonds between Asp-beta 99 and Tyr-alpha 42 and between Asp-beta 99 and Asn-alpha 97 are broken, thus destabilizing the deoxyquaternary structure of these Hbs. A molecular dynamics method has been used to design compensatory amino acid substitutions in these Hbs that can restore their allosteric properties. We have designed a compensatory mutation in a naturally occurring mutant Hb, Hb Kempsey (Asp-beta 99-->Asn), and have produced it using our Escherichia coli expression plasmid pHE2. We have determined the O2 binding properties of this recombinant double mutant Hb, Hb(Asp-beta 99-->Asn and Tyr-alpha 42-->Asp) and have used 1H NMR spectroscopy to investigate the tertiary structures around the heme groups and the quaternary structure in the alpha 1 beta 2 subunit interface. Our results clearly show that the Tyr-alpha 42-->Asp replacement can substantially compensate for the functional defect of Hb Kempsey caused by the Asp-beta 99-->Asn substitution. The structural and functional information derived from this recombinant Hb provides insights into the structural basis of allosterism and the design of compensatory amino acid substitutions to restore the functional properties of other abnormal HBs associated with hemoglobinopathies.

Allosteric Regulation↗

PubMed1994

The effects of phospholipid unsaturation and alcohol perturbation at the protein/lipid interface probed using fluorophore lifetime heterogeneity.

The influence of phospholipid unsaturation and perturbation by alcohols, on the membrane protein/lipid interface, was probed using the fluorescence decay properties of 1,6-diphenyl-1,3,5-hexatriene (DPH) and DPH attached to the sn-2 chain of phosphatidylcholine (DPH-PC), in lipid bilayers and microsomal membranes. With microsomal membranes it was found that it was appropriate to describe the fluorescence decay of DPH-PC as a range of decay rates, accomplished by fitting the data to a bimodal fluorescence lifetime distribution. The major lifetime center had a broad distributional width, indicative of excited state fluorophore heterogeneity. The effect was attributable to protein, and by inference, the protein/lipid interface, since in vesicles made from total microsomal lipids (i.e., without protein) the fluorescence decay was homogeneous. Upon addition of ethanol or hexanol the width of the lifetime distribution of the major lifetime center increased, indicating increased environmental heterogeneity. It was confirmed that the effect was manifest at the protein/lipid interface, and not due to lipid-reorganizational factors, since it could also be obtained using a simple lipid bilayer vesicle system with apocytochrome c as a model membrane protein, and DPH instead of DPH-PC. Environmental heterogeneity was also found to increase with increased phosphatidylcholine (sn-2) unsaturation. The environmental heterogeneity at the protein/lipid interface could arise from a combination of varying polarities of amino acid side chains and of water that may intercalate in packing defects on the hydrophobic surface of the protein. Therefore the results could be explained on the basis of an increased degree of hydration at the protein/lipid interface. Such an effect offers a route whereby acyl chain perturbation and increased unsaturation might influence protein conformation and hence function.

Alcohols↗

PubMed1994

An investigation of the ligand-binding site of the glutamine-binding protein of Escherichia coli using rotational-echo double-resonance NMR.

Glutamine-binding protein (GlnBP) is an essential component of the glutamine transport system in Escherichia coli. Rotational-echo double-resonance (REDOR) solid-state nuclear magnetic resonance (NMR) has been used to determine internuclear distances in the complex of GlnBP and its ligand, L-glutamine. REDOR, combined with strategically placed isotopic labels, is effective in obtaining model-independent internuclear distances and thus detailed structural information on the ligand-binding site of GlnBP. The existence of a single histidine residue (His156) in the binding site has provided an excellent probe for distance measurements between protein and ligand. REDOR distances up to 6.3 A have been observed between 13C labels in L-glutamine and 15N labels in His156. These results have unambiguously determined the ligand orientation with respect to the imidazole ring of His156, which is an important first step in refining the ligand-binding-site model of GlnBP in general. The measured distances were also used as constraints in restrained molecular dynamics calculations of the complex using the unliganded crystal structure of GlnBP as the starting point. The simulations clearly show consistency between calculated distances and those measured by REDOR.

Amino Acid Sequence↗