Biomedical subjects

Y B Lee

Publications and source records attributed to Y B Lee.

At least 19 recordsLinked to original sources

PubMed2007

Influence of soil pH and application rate on the oxidation of calcium sulfite derived from flue gas desulfurization.

Calcium sulfite hemihydrate (CaSO(3).0.5H2O), a common byproduct of coal-fired utilities, is fairly insoluble and can decompose to release toxic SO2 under highly acidic soil conditions; however, it can also oxidize to form gypsum. The objective of this study was to examine the effects of application rate and soil pH on the oxidation of calcium sulfite under laboratory conditions. Oxidation rates measured by release of SO4-S to solution decreased with increasing application rate. Leachate SO4-S from soils amended with 1.0 to 3.0 g kg-1 CaSO3 increased over a 21 to 28 d period before reaching a plateau. At 4 g kg-1, maximum SO4-S release was delayed until Week 7. Oxidation and release of SO4-S from soil amended with 3.0 g kg-1 calcium sulfite increased markedly with decreasing soil pH. After only 3 d incubation, the concentrations of SO4-S in aqueous leachates were 77, 122, 170, 220, and 229 mg L-1 for initial soil pH values of 7.8, 6.5, 5.5, 5.1, and 4.0, respectively. At an initial soil pH value of 4.0, oxidation/dissolution did not increase much after 3 d. At higher pH values, oxidation was maximized after 21 d. These results suggest that autumn surface applications of calcium sulfite in no-till systems should permit ample time for oxidation/dissolution reactions to occur without introducing biocidal effects related to oxygen scavenging. Soil and annual crops can thus benefit from additions of soluble Ca and SO4 if calcium sulfite is applied in advance of spring planting.

Calcium↗

PubMed2006

The mitochondrial genome of the Korean hairstreak, Coreana raphaelis (Lepidoptera: Lycaenidae).

We determined the complete nucleotide sequences of the mitochondrial genome (mitogenome) of the Korean hairstreak, Coreana raphaelis (Lepidoptera: Lycaenidae). The entire mitochondrial DNA (mtDNA) molecule was 15,314 bp long. The C. raphaelis genes were in the same order and orientation as the completely sequenced mitogenomes of other lepidopteran species, except for the presence of an extra copy of tRNA(Ser)(AGN). High similarity in primary sequence and secondary structure between the two tandemly located copies of the tRNA(Ser)(AGN) suggest a recent duplication of an original single tRNA(Ser)(AGN). The DHU arm of the two copies of tRNA(Ser)(AGN) formed a simple loop as seen in many other metazoan mt tRNA(Ser)(AGN). The putative initiation codon for the C. raphaelis COI gene appears to be a tetranucleotide, TTAG, found commonly in the sequenced lepidopterans. ATPase8, ATPase6, ND4L and ND6 genes, which are next to another protein-coding gene at their 3' end all had the sequences potential to form a hairpin structure, suggesting the importance of such a structure for precise cleavage of the mature protein-coding genes.

AT Rich Sequence↗

PubMed2006

Neural substrates, experimental evidences and functional hypothesis of acupuncture mechanisms.

OBJECTIVES: Although acupuncture therapy has demonstrated itself to be effective in several clinical areas, the underlying mechanisms of acupuncture in general and the analgesic effect in particular are, however, still not clearly delineated. We, therefore, have studied acupuncture analgesic effect through fMRI and proposed a hypothesis, based on the obtained result, which will enlighten the central role of the brain in acupuncture therapy. METHODS: The proposed model, termed as a broad sense hypothalamus-pituitary-adrenal (BS-HPA) axis, was based on our observed neuroimaging results. The model incorporates the stress-induced HPA axis model together with neuro-immune interaction including the cholinergic anti-inflammatory model. RESULTS: The obtained results coupled with accumulating evidence suggest that the central nervous system is essential for the processing of these effects via its modulation of the autonomic nervous system, neuroimmune system and hormonal regulation. CONCLUSIONS: Based on our fMRI study, it appears that understanding the effects of acupuncture within a neuroscience-based framework is vital. Further, we have proposed the broad sense-HPA axis hypothesis which incorporates the experimental results.

Acupuncture Therapy↗

PubMed2006

Pharmacokinetics and bioequivalence evaluation of two gabapentin preparations after a single oral dose in healthy Korean volunteers.

OBJECTIVE: To evaluate the bioequivalence of a single oral 400 mg dose of 2 gabapentin preparations in healthy male Korean volunteers. SUBJECTS, MATERIALS AND METHODS: The study was conducted as a randomized, 2-period crossover design in 26 healthy male Korean volunteers who received a single oral dose of 400 mg gabapentin capsule in each study period. There was a 7-day washout period between the doses. Serum concentrations of gabapentin up to 24 hours after the administration were determined using a validated HPLC method with fluorescence detection. In addition, in vitro dissolution profiles of both preparations were examined. The pharmacokinetic parameters such as AUC(0.t) (the area under the curve from zero to the time), AUC(0-infinity) (the area under the curve from zero to infinity), C(max) (maximum serum concentration), t(max) (time to reach C(max)) and t1/2 (terminal half-life) were analyzed by noncompartmental analysis, and the analysis of variance (ANOVA) was carried out using logarithmically transformed AUC(0-t), AUC(0-infinity) and C(max) and untransformed t(max). RESULTS: In vitro dissolution profiles were very similar at all media. There were no significant differences between the two preparations in AUC(0-t), AUC(0-infinity) and C(max). The point estimates (90% confidence intervals) for AUC(0-t), AUC(0-infinity) and C(max) were 1.0319 (0.9142 - 1.1647), 1.0127 (0.8458 - 1.2127) and 0.9796 (0.8670 - 1.1069), respectively, satisfying the bioequivalence criteria of 0.80 - 1.25 as proposed by the US FDA and the Korean legislation. No statistically significant difference was found for tmax and t1/2 values. CONCLUSION: From the results of the present study, it is indicated that the two preparations of gabapentin are bioequivalent and it can be assumed that they are therapeutically equivalent and exchangeable in clinical practice.

Administration, Oral↗

PubMed2004

Pharmacokinetics and bioequivalence evaluation of 2 levosulpiride preparations after a single oral dose in healthy male Korean volunteers.

OBJECTIVE: To evaluate the bioequivalence of a single oral 25 mg dose of 2 levosulpiride preparations in healthy male Korean volunteers. SUBJECTS, MATERIALS AND METHODS: The study was conducted as a randomized, 2-period crossover design in 28 healthy male Korean volunteers who received a single oral dose of 25 mg levosulpiride tablet in each study period. There was a 6-day washout period between the doses. Serum concentrations of levosulpiride up to 36 hours after the administration were determined using a validated HPLC method with fluorescence detection. In addition, in vitro dissolution profiles of both preparations were examined. The pharmacokinetic parameters such as AUC(0-t) (the area under the curve from zero to the time), AUC(0-infinity) (the area under the curve from zero to infinity), Cmax (maximum serum concentration), tmax (time to reach Cmax) and t1/2 (terminal half-life) were analyzed by non-compartmental analysis, and the analysis of variance (ANOVA) was carried out using logarithmically transformed AUC(0-t), AUC(0-infinity) and Cmax, and untransformed Tmax. RESULTS: In vitro dissolution profiles were similar by calculating similarity factor (f2 = 67.73). There were no significant differences between the 2 preparations in AUC(0-t), AUC(0-infinity) and Cmax. The point estimates (90% confidence intervals) for AUC(0-t), AUC(0-infinity) and Cmax were 1.085 (1.003-1.173), 1.069 (0.991-1.153) and 1.075 (0.954 to 1.210), respectively, satisfying the bioequivalence criteria of 0.80-1.25 as proposed by the US FDA and the Korean legislation. No statistically significant difference was found for tmax and t1/2 values. CONCLUSION: From the results of the present study, it is indicated that the 2 preparations of levosulpiride are bioequivalent and it can be assumed that they are therapeutically equivalent and exchangeable in clinical practice.

Administration, Oral↗

PubMed2001

2-Azabicyclo[2.1.1]hexanes. 2. Substitutent effects on the bromine-mediated rearrangement of 2-azabicyclo[2.2.0]hex-5-enes.

Methyl- and phenyl-substituted N-(ethoxycarbonyl)-2-azabicyclo[2.2.0]hex-5-enes 6 have been prepared by photoirradiation of appropriately substituted 1,2-dihydropyridines. Torquoselectivity is observed in the synthesis of the 3-endo-methyl- and 3-endo-phenyl-2-azabicyclo[2.2.0]hexenes 6c-e from 2-methyl- and 2-phenyl-1,2-dihydropyridines 5c-e. Products formed upon addition of bromine to 3-endo-, 4-, and 5-methyl- and 3-endo-phenyl-substituted N-(ethoxycarbonyl)-2-azabicyclo[2.2.0]hex-5-enes 6a-f were substituent dependent. For 6a,b, which lack substituents at C(3) or C(5), mixtures of unrearranged dibromides 8a,b and rearranged dibromides 9a,b were obtained. With the 3-endo-substituents in 6c-e, only rearranged dibromides 9c-e were formed; 5-methyl substitution afforded mainly unrearranged dibromide 8f and some allylic bromide 10. Both unrearranged 5-endo,6-exo-dibromo-2-azabicyclo[2.2.0]hexanes 8 and rearranged 5-anti-6-anti-dibromo-2-azabicyclo[2.1.1]hexanes 9 are formed stereoselectively. The dibromoazabicyclo[2.1.1]hexanes 9 have been reductively debrominated to afford the first reported 2-azabicyclo[2.1.1]hexanes 11 with alkyl or aryl substituents at C-3.

Bridged Bicyclo Compounds, Heterocyclic↗

PubMed2001

GT1b ganglioside induces death of dopaminergic neurons in rat mesencephalic cultures.

We examined neurotoxicity of GT1b against dopaminergic neurons in vitro. Cultures of mesencephalic cells deprived of serum underwent the loss of 19% of tyrosine hydroxylase immunopositive (TH-ip) neurons. In cultures deprived of serum, treatment with 10-30 microg/ml GT1b attenuated the number of TH-ip neurons by 26-69%, respectively, compared to non-treated cultures. Intriguingly, cultures deprived of serum were more vulnerable to GT1b-induced neurotoxicity. Application of 60 microg/ml GT1b to cultures grown in serum containing media resulted in the loss of 26% of TH-ip neurons, similar to that (28%) observed in serum-deprived cultures treated with 10 microg/ml GT1b. Moreover, in our cultures, absence of nitric oxide (NO) production after GT1b treatment was obvious. The present results strongly suggest direct neurotoxic actions of GT1b against dopaminergic neurons regardless of NO.

Animals↗

PubMed2001

Effects of neurotoxin 6-aminonicotinamide on levels of enzyme activities and metabolites in quail plasma.

Effects of 6-aminonicotinamide (6-AN) on the levels of proteins, metabolites and enzyme activities in the plasma of Japanese quail were investigated. The concentrations of soluble proteins in the pectoral and hindlimb muscle of the 6-AN treated and the pair-fed groups were significantly reduced compared to the control group. In the plasma, the levels of total proteins and albumin were not affected, but the levels of globulin were significantly lower than those of the control and pair-fed groups. In contrast, the levels of glucose and creatine were significantly elevated. Cellulose acetate gel electrophoresis showed that 6-AN induced a new synthesis of prealbumin and also increased the levels of beta-globulin relative to the control and pair-fed groups. In contrast, the levels of gamma-globulin were markedly lower than those of the control group, whereas the levels of alpha-globulin were not affected. The specific activity of alkaline phosphatase of the 6-AN group was significantly lower than that of the control and pair-fed groups and that of aspartate aminotransferase only lower than that of the control group but not the pair-fed group. The specific activities of creatine phosphokinase and lactate dehydrogenase of the 6-AN group were the greatest among the three groups, whereas those of the pair-fed group were greater than those of the control group. The results suggest that 6-AN may interfere with the proper maintenance of energy charges and the immune system function.

6-Aminonicotinamide↗

PubMed2001

Chronological changes of N-methyl-D-aspartate receptors and excitatory amino acid carrier 1 immunoreactivities in CA1 area and subiculum after transient forebrain ischemia.

We investigated changes of immunoreactivities of N-methyl-D-aspartate receptor (NR) and of excitatory amino acid carrier 1 (EAAC-1), the neuronal glutamate transporter, in the vulnerable CA1 area and the less vulnerable subiculum of the gerbil hippocampus at various times following transient forebrain ischemia. At 30 min after ischemia-reperfusion, the intensity of NR immunoreactivity increased markedly in neurons of CA1 and subiculum, particularly NR2A/B, while EAAC-1 immunoreactivity was reduced in CA1. At 3 hr after reperfusion, the density of NR1 immunoreactivity markedly decreased in CA1. In contrast EAAC-1 immunoreactivity increased in CA1 and in the subiculum. At 12 hr after reperfusion, the decrease of NR1 immunoreactivity was not detected whereas EAAC-1 immunoreactivities in the CA1 area were intensified. In the subiculum, both NR subunits immunoreactivities decreased significantly, in contrast to the maintenance of EAAC-1 immunoreactivity. At 24 hr after reperfusion, both NR2A/B and EAAC-1 immunoreactivities decreased markedly in CA1 and subiculum. We tentatively suggest that the increase of NR immunoreactivity in CA1 at early times after ischemia-reperfusion may increase the delayed neuronal death, and that the increase or maintenance of EAAC-1 immunoreactivity at early times after ischemia-reperfusion may be an important factor in survival of neurons.

Amino Acid Transport System X-AG↗

PubMed2001

The effects of famciclovir and epidural block in the treatment of herpes zoster.

In our previous study, we concluded that an epidural blockade combined with intravenous acyclovir is very effective in treating the acute pain in herpes zoster and postherpetic neuralgia. We evaluated the efficacy of oral famciclovir and epidural blockade on the pain of herpes zoster, compared to acyclovir administered intravenously and epidural blockade. For this purpose, we examined a new group treated with famciclovir and epidural blockade to compare with the group treated with acyclovir and epidural blockade in our previously study. The changes in the intensity of pain, the number of days required for relief of pain, and the total duration of pain were checked. We compared the days required for relief of pain (DRP) and the total duration of pain (TDP) of this group with those of the previous studied group treated with acyclovir and epidural blockade. DRP was significantly less, but TDP was similar. DRP and TDP were significantly lower, if the patients were treated within 7 days of symptom onset. The patients had a shorter DRP regardless of pain type than the previously studied group treated with acycolvir and epidural blockade. For the severe and moderate pain grades, there was a shorter DRP from 100 to 10. TDP was not significantly different for the groups regardless of pain type or grade. We believe that famciclovir and epidural blockade are very effective in treating the pain of herpes zoster, with a view to shortening the period of acute pain, providing similar effects on the prevention of postherpetic neuralgia, and being convenient to administer, compared to intravenous acyclovir and epidural blockade in our previous study.

2-Aminopurine↗

PubMed2001

Depletion of mitochondrial DNA alters glucose metabolism in SK-Hep1 cells.

Maternally inherited mitochondrial DNA (mtDNA) has been suggested to be a genetic factor for diabetes. Reports have shown a decrease of mtDNA content in tissues of diabetic patients. We investigated the effects of mtDNA depletion on glucose metabolism by use of rho(0) SK-Hep1 human hepatoma cells, whose mtDNA was depleted by long-term exposure to ethidium bromide. The rho(0) cells failed to hyperpolarize mitochondrial membrane potential in response to glucose stimulation. Intracellular ATP content, glucose-stimulated ATP production, glucose uptake, steady-state mRNA and protein levels of glucose transporters, and cellular activities of glucose-metabolizing enzymes were decreased in rho(0) cells compared with parental rho(+) cells. Our results suggest that the quantitative reduction of mtDNA may suppress the expression of nuclear DNA-encoded glucose transporters and enzymes of glucose metabolism. Thus this may lead to diabetic status, such as decreased ATP production and glucose utilization.

Adenosine Triphosphate↗

PubMed2000

Rapid increase in immunoreactivity to GFAP in astrocytes in vitro induced by acidic pH is mediated by calcium influx and calpain I.

In higher vertebrates, reactive gliosis resulting from injury to the central nervous system (CNS) is characterized by a rapid increase in immunoreactivity (IR) to glial fibrillary acidic protein (GFAP). Little is known about the extracellular signals that initiate the increase in GFAP-IR following CNS injury. We demonstrated recently [T.H. Oh, G.J. Markelonis, J.R. Von Visger, B. Baik, M.T. Shipley, Acidic pH rapidly increases immunoreactivity of glial fibrillary acidic protein in cultured astrocytes, Glia 13 (1995) 319-322] that a rapid increase in GFAP-IR can be evoked in mature astrocyte cultures by exposing the cells to an acidic medium. We investigated the intracellular pathway(s) involved in initiating increased GFAP-IR, a hallmark of reactive astrocytes. The increase in GFAP-IR produced by exposure to acidic medium was blocked by pretreatment with nickel ions, by such blockers of L-type calcium channels as nifedipine, verapamil and diltiazem, by calpain inhibitor I, or by the intracellular calcium chelator, BAPTA-AM. At physiological pH, treatment with the calcium ionophore, A23187, resulted in increased GFAP-IR which could be blocked by pretreatment with calpain inhibitor I. Astrocytes exposed to low pH exhibited a marked increase in a GFAP fragment with a molecular weight of 48 kDa. In astrocytes exposed to acidic medium, alpha-fodrin, a selective endogenous substrate of calpain, was also found to be hydrolyzed producing fragments with molecular weights of 120-150 kDa. As anticipated, pretreatment with calpain inhibitor I prevented the proteolytic degradation of both GFAP and alpha-fodrin in these samples. These results suggest that the initial increase in GFAP-IR after CNS injury appears to be linked to Ca(++) influx, and is mediated further by a proteolytic process that seemingly involves the activation of the calcium-dependent protease, calpain I.

Acids↗

PubMed2000

Acute application of interleukin-1beta induces Ca(2+) responses in human microglia.

The effects of the pro-inflammatory cytokine interleukin-1-beta (IL-1beta) on levels of intracellular calcium [Ca(2+)](i) in cultured human microglia have been studied using the fluorescent Ca(2+) indicator fura-2. IL-1beta (2 ng/ml) caused a slow, progressive increase in [Ca(2+)](i) in standard Ca(2+)-containing physiological solution (PSS). A similar effect was observed in separate studies using Ca(2+)-free PSS, however, the mean rate of increase was significantly lower than that measured with PSS. Similar results were obtained in a separate protocol, where cells were exposed to both IL-1beta in Ca(2+)-free PSS and PSS. The slope of the IL-1beta induced increase of [Ca(2+)](i) in Ca(2+)-free PSS was not altered when adenosine triphosphate was added prior to application of the cytokine. These results suggest that IL-1beta-induced responses in human microglia involve both a Ca(2+) entry pathway and a mechanism of intracellular increase other than from IP(3)-sensitive stores.

Calcium↗

PubMed2000

p38 map kinase regulates TNF-alpha production in human astrocytes and microglia by multiple mechanisms.

In the vertebrate central nervous system (CNS), tumour necrosis factor-alpha (TNF-alpha) is produced by astrocytes and microglia and mediates cell injury in nerve cells and oligodendrocytes. In the present study, we have used a specific inhibitor of p38 MAP kinase, SB203580 to examine the role of p38 MAP kinase in regulation of TNF-alpha production in human astrocytes and microglia in terms of levels of mRNA and secreted protein. A reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that increased levels of TNF-alpha mRNA were induced in astrocytes by IL-1beta treatment, and in microglia by bacterial lipopolysaccharide (LPS). In microglia, treatment with SB203580 reduced the level of TNF-alpha mRNA, but in astrocytes it did not. However, the secretion of TNF-alpha by both astrocytes and microglia was markedly inhibited by SB203580 at a low concentration. TNF-alpha secretion was reduced approximately 80% in astrocytes and 85% in microglia. The results demonstrate a key role played by p38 MAP kinase in upregulation of TNF-alpha mRNA levels in LPS-activated human microglia, whereas p38 MAP kinase is involved in post-transcriptional regulation of TNF-alpha production at translational level in IL-1beta-activated human astrocytes.

Astrocytes↗

PubMed2000

Role of tumor necrosis factor-alpha in neuronal and glial apoptosis after spinal cord injury.

We investigated the role of tumor necrosis factor (TNF)-alpha in the onset of neuronal and glial apoptosis after traumatic spinal cord crush injury in rats. A few TUNEL-positive cells were first observed within and surrounding the lesion area 4 h after injury, with the largest number observed 24-48 h after injury. Double-labeling of cells using cell type-specific markers revealed that TUNEL-positive cells were either neurons or oligodendrocytes. One hour after injury, an intense immunoreactivity to TNF-alpha was observed in neurons and glial cells in the lesion area, but also seen in cells several mm from the lesion site rostrally and caudally. The level of nitric oxide (NO) also significantly increased in the spinal cord 4 h after injury. The injection of a neutralizing antibody against TNF-alpha into the lesion site several min after injury significantly reduced both the level of NO observed 4 h thereafter as well as the number of apoptotic cells observed 24 h after spinal cord trauma. An inhibitor of nitric oxide synthase (NOS), N(G)-monomethyl-l-arginine acetate (l-NMMA), also reduced the number of apoptotic cells. This reduction of apoptotic cells was associated with a decrease in DNA laddering on agarose gel electrophoresis. These results suggest that: (i) TNF-alpha may function as an external signal initiating apoptosis in neurons and oligodendrocytes after spinal cord injury; and (ii) TNF-alpha-initiated apoptosis may be mediated in part by NO as produced by a NOS expressed in response to TNF-alpha.

Animals↗

PubMed2000

Effect of S-adenosylmethionine on hepatic injury from sequential cold and warm ischemia.

We investigated whether S-adenosylmethionine (SAM) treatment improved ischemic injury using perfused rat liver after sequential periods of 24 h cold and 20 min re-warming ischemia. SAM (100 micromol/L) was added to University of Wisconsin (UW) solution and Ringers lactate solution. After cold and sequential warm ischemia, releases of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP) markedly increased during reperfusion. The increase in PNP was significantly reduced by SAM treatment. While the concentration of reduced glutathione (GSH) in ischemic livers significantly decreased, the concentration of glutathione disulfide (GSSG) increased. This decrease in GSH and increase in GSSG were suppressed by SAM treatment. Lipid peroxidation was elevated in cold and warm ischemic and reperfused livers, but this elevation was also prevented by SAM treatment. Hepatic ATP levels were decreased in the ischemic and reperfused livers to 42% of the control levels. However, treatment with SAM resulted in significantly higher ATP levels and preserved the concentration of AMP in ischemic livers. Our findings suggest that SAM prevents oxidative stress and lipid peroxidation and helps preserve hepatic energy metabolism.

Adenosine Triphosphate↗

PubMed2000

Effect of dietary linoleate/alpha-linolenate balance on the brain lipid composition, reproductive outcome and behavior of rats during their prenatal and postnatal development.

The effect of the dietary linoleate (LA)/alpha-linolenate (LNA) balance during development on the brain lipid composition, reproductive outcome and behavior of rats was studied. Female rats were fed on experimental diets during pregnancy and the resulting pups for 16 weeks. The dietary LA/LNA ratios were 1.07 (LA1), 2.64 (LA2), 4.45 (LA3), 7.68 (LA4) and 10.35 (LA5). The relative content of docosahexaenoate (DHA) in the brain of pups tended to increase with decreasing LA/LNA ratio at 0 and 3 weeks, while the level of DHA was maintained constant at 16 weeks regardless of the dietary LA/LNA ratio. The learning ability was measured at 12 weeks of age, and there was no difference among the groups. In an open field test, the exploratory index was significantly lower in the LA1 group than in the LA2 group. The LA1 group had a smaller litter size and lower survival rate than the other groups. We conclude that if the diet contained appropriate amounts and balance of LA and LNA, it was possible for rats to synthesize an appropriate amount of DHA and have normal behavioral activity without DHA supplementation.

Animals↗

PubMed1999

Complex formation between deoxyhypusine synthase and its protein substrate, the eukaryotic translation initiation factor 5A (eIF5A) precursor.

Deoxyhypusine synthase catalyses the first step in the post-translational synthesis of hypusine [Nepsilon-(4-amino-2-hydroxybutyl) lysine] in a single cellular protein, the precursor of eukaryotic initiation factor 5A (eIF5A). Deoxyhypusine synthase exists as a tetramer with four potential active sites. The formation of a stable complex between human deoxyhypusine synthase and its protein substrate, human recombinant eIF5A precursor (ec-eIF5A), was examined by affinity chromatography using polyhistidine-tagged (His.Tag) ec-eIF5A, by a gel mobility-shift method, and by analytical ultracentrifugation. Deoxyhypusine synthase was selectively retained by His.Tag-ec-eIF5A immobilized on a resin. The complex of deoxyhypusine synthase and ec-eIF5A was separated from the free enzyme and protein substrate by electrophoresis under non-denaturing conditions. The stoichiometry of the two components in the complex was estimated to be 1 deoxyhypusine synthase tetramer to 1 ec-eIF5A monomer by N-terminal amino acid sequencing of the complex. Equilibrium ultracentrifugation data further supported this 1:1 ratio and indicated a very strong interaction of the enzyme with ec-eIF5A (Kd</=0.5 nM). Formation of the complex was not dependent on NAD+ or spermidine and occurred at pH7.0-9.2. An enzyme-product complex, as well as the deoxyhypusine-containing product (modified ec-eIF5A), was also detected at pH7.0-9.2 in a complete reaction mixture containing 1 mM spermidine.

Cell Extracts↗