Biomedical subjects

L S Chang

Publications and source records attributed to L S Chang.

At least 55 records · Page 3Linked to original sources

PubMed1998

Unfolding/folding studies on cobrotoxin from Taiwan cobra venom: pH and GSH/GSSG govern disulfide isomerization at the C-terminus.

Refolding of cobrotoxin was assessed by the exposure degree of its single Trp determined by an acrylamide quenching study. The change in the accessibility of Trp for acrylamide quantitatively reflected the formation of folded cobrotoxin, and the data were confirmed by HPLC and gel electrophoresis analyses. However, the site-specific information provided by quenching Trp fluorescence revealed that the ordered structure in the neighborhood of Trp was attained prior to the complete formation of the tertiary structure of cobrotoxin. HPLC analyses showed that, in addition to refolded cobrotoxin, two novel species (cobrotoxin II and cobrotoxin III) with isomerization of disulfide bonds at the C-terminus of the toxin molecule were produced along the folding reaction. The disulfide pairings in cobrotoxin II and cobrotoxin III were Cys43-Cys55 and Cys54-Cys60 and Cys43-Cys60 and Cys54-Cys55, respectively. Among the three possible two-disulfide species at the C-terminus, the disulfide linkages Cys43-Cys60 and Cys54-Cys55 of cobrotoxin III caused a marked decrease in lethality and resulted in a conformation which was notably different from that observed with the native toxin molecule as evidenced by CD spectra. The refolding reaction was accelerated by the addition of GSH/GSSG, and the resulting products were mostly folded cobrotoxin. However, if GSH/GSSG was not added into the initial folding materials, the yields of cobrotoxin II and cobrotoxin III greatly increased. The conversion of cobrotoxin to its isomers was to be irreversible and pH-dependent: the higher the pH, the faster the rate of conversion. However, this conversion could be partly inhibited by GSH/GSSG. Cobrotoxin II and cobrotoxin III were purified from Taiwan cobra venom as well, and their yields in comparison to that of cobrotoxin in venom were similar to that noted with the folded products in the presence of GSH/GSSG. Moreover, the rate of disulfide isomerization was expected to be slow in venom fluid in which the pH was approximately pH 6.2. Thus, the finding that cobrotoxin represents the predominant neurotoxin species in Taiwan cobra venom is probably associated with the synergistic effects of GSH/GSSG and pH.

Amino Acid Sequence↗

PubMed1998

Differentially expressed snoRNAs in Bungarus multicinctus (Taiwan banded krait).

Twenty novel snoRNAs forming extensive sequence complementarities to mature 5S rRNA were identified from Bungarus multicinctus by reverse transcription-polymerase chain reaction. It was found that the snoRNA species were differentially transcribed in different tissues as evidenced by single stranded conformational polymorphism analysis and direct nucleotide sequence analysis. Although the diversity in the sequences of snoRNAs is observed, comparison of these snoRNA genes reveals that the regions involved in binding to 5S rRNA are highly conserved and form two 12-nt-15-nt tracts of complementarity to phylogenetically invariant sequences in eukaryotic 5S rRNAs. Nevertheless, the lower conservation of box C/D or box H/ACA in these snoRNAs was observed. Likewise, the sequences in several fish and human genes forming perfect duplexes with 5S rRNA also did not highly retain these box elements. These results may infer that the box elements are dispensable for the function of snoRNA species identified in the present study. Moreover, the novel finding of the differentially expressed snoRNA variants in B. multicinctus suggests that the snoRNA genes are selectively processed in different tissues and are likely associated with tissue-specific regulation of their host gene transcripts.

Animals↗

PubMed1998

Developmental expression of mouse erythrocyte protein 4.2 mRNA: evidence for specific expression in erythroid cells.

Erythrocyte protein 4.2 (P4.2) is an important component of the erythrocyte membrane skeletal network with an undefined biologic function. Presently, very little is known about the expression of the P4.2 gene during mouse embryonic development and in adult animals. By using the Northern blot and in situ hybridization techniques, we have examined the spatial and temporal expression of the P4.2 gene during mouse development. We show that expression of the mouse P4.2 gene is temporally regulated during embryogenesis and that the P4.2 mRNA expression pattern coincides with the timing of erythropoietic activity in hematopoietic organs. P4.2 transcripts are first detected in embryos on day 7.5 of gestation and are localized exclusively in primitive erythroid cells of yolk sac origin. These erythroid cells remain to be the only source for P4.2 expression until the switch of the hematopoietic producing site to fetal liver. In mid- and late-gestation periods, P4.2 mRNA expression is restricted to the erythroid cells in fetal liver and to circulating erythrocytes. Around and after birth, the site for P4.2 expression is switched from liver to spleen and bone marrow, and P4.2 transcripts are only detected in cells of the erythroid lineage. These results provide the evidence for specific P4.2 expression in erythroid cells. In addition, the timing and pattern of expression of the P4.2 gene suggest the specific regulation of the P4.2 gene.

Animals↗

PubMed1998

Expression of osteopontin mRNA in normal and stone-forming rat kidney.

Human urine contains several macromolecules which inhibit calcium oxalate crystallization. Osteopontin (or uropontin), a secreted phosphoglycoprotein with the amino acid sequence Arg-Gly-Asp (RGD) and high affinity to hydroxyapatite, is one such inhibitor. To investigate the action of this protein on renal stone formation, the expression osteopontin gene in normal and chemically induced urolithiasis rat kidney was compared at both mRNA and protein levels. Northern blot analysis shown a significant increase of osteopontin mRNA level in stone-forming rat kidney compared with normal ones. In an in situ hybridization study, we localized the transcripts of the osteopontin gene in epithelial cells of both distal and collective tubules, and found a remarkably strong signal in stone-forming rats. The amount and distribution of the protein in kidney from immunocytochemistry staining showed the same pattern as seen in situ hybridization. These findings indicate that osteopontin may be an important macromolecule in the normal endogenous defence against the formation of urinary calculi.

Animals↗

PubMed1998

Structural factors affect the interactions of anticardiotoxin antibodies and cobra venom cardiotoxins.

Two antibody preparations against cardiotoxins were raised by immunizing rabbits with cardiotoxin 1 and cardiotoxin 3, respectively. The two antibody preparations showed precipitin reactions with cardiotoxins 1, 2, 3 and 5, respectively. However, the results of competitive enzyme-linked immunoassay revealed that the respective cardiotoxin molecules exhibited different reactivity toward anticardiotoxin antibodies. Moreover, the order of reactivity with antibodies was not in line with the degree of their sequence identity. This suggest that the anticardiotoxin antibodies may recognize conformational epitopes rather than sequential ones in the toxin molecules. Alternatively, the four cardiotoxins reacted well with the antibodies in the absence of competitor, suggesting that sequence variations with cardiotoxin molecules may not exclusively influence the potential use of the anticardiotoxin antibodies for the neutralization of the activity of cardiotoxin variants.

Amino Acid Sequence↗

PubMed1998

Characterization and cloning of long neurotoxin homolog from Naja naja atra.

The cDNA encoding a long neurotoxin homolog was constructed from the cellular RNA isolated fom the venom glands of Naja naja atra (Taiwan cobra) by reverse transcription-polymerase chain reaction. BLAST searches for sequence similarity in the GenBank databases reveal that the cDNA sequence of the long neurotoxin homolog is not highly homologous with long and short neurotoxins. Although the long neurotoxin homolog exhibited an activity to inhibit acetylcholine-induced muscle contractions as Naja naja atra cobrotoxin, the degree of inhibition caused by the addition of long neurotoxin homolog was only approximately 35% of that observed with the addition of cobrotoxin. Moreover, the primary structure of the long neurotoxin homolog did not fulfill the characteristic features of long or short neurotoxins. Together with long neurotoxin homologs from other snake species, they probably represent an evolutionary divergence between long and short neurotoxins.

Acetylcholine↗

PubMed1998

Identification of arg-30 as the essential residue for the enzymatic activity of Taiwan cobra phospholipase A2.

Taiwan cobra (Naja naja atra) phospholipase A2 (PLA2) was inactivated by arginine-specific reagents, phenylglyoxal and 1, 2-cyclohexanedione. Kinetic analyses of the modification reaction revealed that the inactivation of PLA2 followed pseudo-first-order kinetics and the loss of activity was correlated with the incorporation of one molecule of modification reagent per PLA2 molecule. This was confirmed by the results of amino acid composition determination, that showed that a marked decrease in enzymatic activity was associated with the modification of one arginine residue. Tryptic cleavage of the modified protein and microsequencing revealed that Arg-30 was the functionally essential residue. The incorporation of a modifier into the PLA2 did not significantly affect the secondary structure of the enzyme, as revealed by the CD spectrum, and Ca2+-binding of the modified PLA2 was unaffected. Nevertheless, the nonpolarity of the active site of PLA2 markedly decreased with the arginine modification, as evidenced by the decreases in the enhancement of Trp and 8-anilinonaphthalene sulfonate fluorescence. These results, together with those of X-ray crystallographic analysis of N. naja atra PLA2 [Scott et al. (1990) Science 250, 1541-1546], demonstrate that Arg-30 is one of the residues involved in the interfacial binding of a PLA2 molecule with its substrate.

Animals↗

PubMed1998

Effects of outlet obstruction and its reversal on mitochondrial enzyme activity in rabbit urinary bladders.

PURPOSE: This study investigated the effects of outlet obstruction and its reversal on mitochondrial enzyme activity in the rabbit urinary bladder. MATERIALS AND METHODS: We induced mild bladder outlet obstruction in male New Zealand White rabbits. Following two weeks of obstruction, one group of animals (n = 6) was sacrificed, while outlet obstruction was relieved in three additional groups of animals, which were sacrificed one (n = 5), two (n = 5) and four (n = 5) weeks after relieving the obstruction. Seven sham operated rabbits served as controls. We extracted mitochondria from fresh detrusor and assayed activities of key mitochondrial enzymes in the citric acid cycle, citrate synthase and malate dehydrogenase, as well as those in the electron transport chain, succinate cytochrome c reductase, NADH-cytochrome c reductase and cytochrome c oxidase. With high performance liquid chromatography (HPLC) we determined the tissue content of phosphocreatine and the adenine nucleotides (ATP, ADP and AMP), which was used for calculating energy charge. Responses of detrusor strips to 500 microM bethanechol and 120 mM KCl provided the assessment of detrusor contractility. RESULTS: Contractile response of the detrusor strips to bethanechol stimulation was significantly reduced by outlet obstruction, nevertheless, it recovered gradually toward the control level after the relief of outlet obstruction. Outlet obstruction reduced the detrusor content of phosphocreatine, ATP and energy charge. After relieving the obstruction, however, these recovered gradually, reaching control levels 4 weeks later. The activities of all assayed enzymes were reduced by two weeks of outlet obstruction. Relieving the obstruction restored enzyme activity gradually but at different rates for different enzymes. Activities of the citric acid cycle enzymes citrate synthase and malate dehydrogenase recovered and were similar to control levels four weeks after relief of the obstruction. Of the enzymes in the electron transport chain, NADH cytochrome reductase activity recovered most quickly by one week after relief of the obstruction. The activity of cytochrome c oxidase improved more slowly, but 4 weeks after relieving the obstruction it, also, was restored and was similar to the control level. Succinate cytochrome reductase activity remained lower than the control over the entire four weeks of recovery. CONCLUSIONS: The close association between mitochondrial enzyme activity, energy metabolism and contractility of the detrusor indicates the important role of mitochondrial enzyme damage in decreasing energy production and impairing contractile function of the urinary bladders following outlet obstruction. Our findings also show that various mitochondrial enzymes exhibit different susceptibilities and reversibilities to pathological stress.

Adenine Nucleotides↗

PubMed1998

Recovery of microvascular blood perfusion and energy metabolism of the obstructed rabbit urinary bladder after relieving outlet obstruction.

We investigated the changes in urinary bladder microvascular blood perfusion and energy metabolism following outlet obstruction and after relieving the obstruction. We induced mild bladder outlet obstruction in male New Zealand White rabbits. Following 2 weeks of obstruction, one group of animals (n = 6) was sacrificed, while outlet obstruction was relieved in three additional groups, which were sacrificed 1 (n = 5), 2 (n = 5) and 4 (n = 5) weeks after relieving the obstruction. Seven sham-operated rabbits served as controls. Before obstruction, immediately before relieving the obstruction and preceding the sacrifice, the microvascular blood perfusion of the urinary bladder was measured using a laser Doppler blood flowmeter. The detrusor content of phosphocreatine and adenine nucleotides was determined by high-performance liquid chromatography. The results showed: (1) 2 weeks of outlet obstruction significantly decreased the bladder microvascular blood perfusion, which recovered gradually after relief of the obstruction and returned to the control level by 4 weeks of obstruction reversal; (2) outlet obstruction reduced detrusor energy charge and phosphocreatine content, which were restored in parallel after relieving the obstruction; by 4 weeks the bladder had regained their normal energy producing capability; (3) bladder microvascular perfusion has a very close correlation with detrusor energy charge (r = 0.791, p < 0.001). In conclusion, our findings of the close correlation between microvascular perfusion and energy production in bladder outlet obstruction suggest an important role for the decreased microvascular blood perfusion in reducing bladder energy production.

Adenine Nucleotides↗

PubMed1998

Complicated urinary tract infection: analysis of 179 patients.

BACKGROUND: The aim of our study was to investigate the incidence, bacteriology, management and outcome of complicated urinary tract infections (UTIs) at the Veterans General Hospital-Taipei. METHODS: Between June, 1993, and July, 1994, medical records of 2,566 patients admitted to the Division of Urology, Veterans General Hospital-Taipei, were retrospectively reviewed. Of these patient, 1,322 had a diagnosis of benign prostatic hyperplasia (BPH), 607 were admitted for renal stones, 496 for ureteral stones, 75 for transitional cell carcinoma (TCC) of the urinary bladder, 47 for renal tumors and 19 for TCC of the ureter. Among all patients studied, 179 (6.98%) acquired a complicated UTI. Of these, 81 were admitted for BPH, 46 for renal stones, 42 for ureteral stones, five for TCC of the urinary bladder, three for renal tumors and two for TCC of the ureter. RESULTS: Of the 179 patients with complicated UTIs, 155 were men and 24 were women. The urine culture positive rate was 76.0% (136/179) and the most common bacteria were Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa. The principle mode of treatment included parenteral antibiotics and urinary diversion (percutaneous nephrostomy and Foley catheterization), when necessary. The infection control rate for these complicated UTIs was 96.3% for BPH, 95.5% for renal stone, 97.6% for ureteral stone, 80% for TCC of the urinary bladder, 100% for renal tumor and 100% for TCC of the ureter. Mortality due to complicated UTI was 3.9% (7/179). CONCLUSIONS: We concluded that the prognosis of complicated UTI is good if diagnosis and appropriate treatment are given promptly. Early drainage to relieve obstruction and intravenous antibiotics are initially necessary. Surgical intervention is required to resolve functional or structural abnormalities after the UTI has been controlled.

Adult↗

PubMed1998

Successful treatment of a bulky seminoma in an abdominal undescended testis: a case report.

An undescended testis is a major risk factor for the development of testicular malignancy. Herein we report the case of a 40-year-old man with a bulky seminoma arising in an uncorrected abdominal undescended testis. The definitive diagnosis was established by results of sonoguided biopsy and tumor marker studies. The patient was successfully treated with initial cisplatin and etoposide combination chemotherapy followed by resection of the residual tumor. The optimal diagnosis and treatment of this rare disease is discussed.

Adult↗

PubMed1997

Genomic structures of cardiotoxin 4 and cobrotoxin from Naja naja atra (Taiwan cobra).

Two genomic DNAs with the size of 2.3 kb and 2.4 kb, which were isolated from the liver of Naja naja atra (Taiwan cobra), encoded the precursors of cardiotoxin 4 and cobrotoxin, respectively. Both genes shared virtually identical overall organization with three exons separated by two introns, which were inserted in the similar positions of the gene's coding regions. Moreover, their nucleotide sequences shared approximately 84.2% identity. This result reveals the evolutionary relationship between cardiotoxin and cobrotoxin. The exon/intron structures of cardiotoxin 4 and cobrotoxin genes were similar to that reported for erabutoxin c gene, a neurotoxin genomic DNA from a sea snake (Laticauda semifasciata). However, in contrast to the finding that the intron 2 of these genes had a similar size, a notable variation with the size of intron 1 was observed (1233 bp, 1269 bp and 197 bp for cardiotoxin 4, cobrotoxin and erabutoxin c genes, respectively). The different size with intron 1 is due to the middle region at the first intron of cardiotoxin 4 and cobrotoxin genes, which encoded small nucleolar RNA (snoRNA), being absent in that of erabutoxin c gene. These results, together with the finding of the potential mobility of snoRNA genes during evolution, suggest that intron insertions or deletions of snoRNA genes occur with the evolutionary divergence of snake neurotoxins and cardiotoxins.

Amino Acid Sequence↗

PubMed1997

Inhibition of mouse thymidylate synthase promoter activity by the wild-type p53 tumor suppressor protein.

The p53 tumor suppressor protein is an important negative regulator of the G1 to S transition in mammalian cells. We have investigated the effect of p53 on the expression of the mouse thymidylate synthase (TS) gene, which normally increases as cells enter S phase. A luciferase indicator gene that was driven by the wild-type or various modified forms of the TATA-less mouse TS promoter was transiently cotransfected with a p53 expression plasmid into TS-deficient hamster V79 cells and the level of luciferase activity was determined. We found that wild-type p53 inhibited TS promoter activity by greater than 95% but had a strong stimulatory effect on an artificial promoter that contained multiple p53-binding sites. In contrast, an expression plasmid that encodes a mutant form of p53 or a wild-type retinoblastoma tumor suppressor protein had little effect on TS promoter activity. Deletion of sequences upstream or downstream of the TS essential promoter region, or inactivation of each of the known elements within the essential promoter region, had no effect on the ability of wild-type p53 to inhibit TS promoter activity. Our observations indicate that the inhibition of TS promoter activity by p53 is not due to the presence of a specific p53 negative response element in the TS promoter. Rather, it appears that p53 inhibits the TS promoter by sequestering ("squelching") one or more general transcription factors.

Animals↗

PubMed1997

The human POLD1 gene. Identification of an upstream activator sequence, activation by Sp1 and Sp3, and cell cycle regulation.

The promoter of the human POLD1 gene encoding the catalytic subunit of DNA polymerase delta is G/C-rich and does not contain a TATA box. Transient transfection analysis in HeLa cells employing POLD1-luciferase chimeric plasmids revealed a core promoter region extending 328 base pairs (bp) from the major transcription initiation site. Multiple elements in this region including two 11-bp direct repeats located between nucleotide positions -92 and -22, play an important role in POLD1 promoter activity. Deletion or linker-replacement mutations of the repeats drastically reduced the promoter activity. A 70-bp DNA fragment containing the two repeats could stimulate the expression of the POLD1 or a heterologous promoter in an orientation-independent manner. DNase I footprinting and band-shift assays showed that HeLa nuclear extracts contained proteins specifically binding to the repeat sequences. Southwestern blot and UV cross-linking analyses identified Sp1 and two 85-kDa proteins that bound to the repeats. Additionally, screening of HeLa cDNA expression libraries for the sequence-specific DNA-binding protein using the 11-bp repeat sequences as the probe, identified a cDNA that corresponds to Sp3, a member of the Sp1 family. Cotransfection studies in Drosophila SL2 cells showed that both Sp1 and Sp3, but not Sp2, could activate the POLD1 promoter through the repeat sequences. The POLD1 promoter activity was induced about 4-fold at the late G1/S boundary in serum-stimulated cells. The 11-bp repeats together with an E2F-like sequence, located adjacent to the major transcription initiation site, were important for the stimulation. Taken together, this study provides a direct evidence for transcriptional regulation of the human POLD1 gene.

Base Sequence↗

PubMed1997

Paraneoplastic elevation of serum alkaline phosphatase in renal cell carcinoma: incidence and implication on prognosis.

PURPOSE: We investigated the incidence and prognostic significance of paraneoplastic elevation of serum alkaline phosphatase in patients with renal cell carcinoma. MATERIALS AND METHODS: Clinical data of 365 pathologically proved renal cell carcinoma cases were reviewed. Serum alkaline phosphatase level greater than 100 units per 1., but without obvious conditions that may cause phosphatase elevation, including metastasis to or disease of liver or bone and pregnancy, was regarded as paraneoplastic serum alkaline phosphatase elevation. Survival was evaluated using the Kaplan-Meier method. RESULTS: Of 365 patients 77 (21.1%) had paraneoplastic serum alkaline phosphatase elevation. The respective incidence from stage I to IV cases was 9.9% (16 of 161), 31.9% (15 of 47), 34.3% (23 of 67) and 25.6% (23 of 90). Patients with stage I disease had the lowest incidence but there were no statistically significant differences among stages II, III and IV disease. Of 77 patients with elevated serum alkaline phosphatase 48 had additional paraneoplastic manifestations. The disease specific 5-year survival rate in patients with normal serum alkaline phosphatase was significantly better than in patients with isolated phosphatase elevation, which in turn was better than in patients with multiple paraneoplastic syndromes (70.7 versus 50.5 versus 30.8%). Patients with persistent or recurrent elevation of serum alkaline phosphatase after radical nephrectomy had metastatic lesion or local recurrence. In some patients serum alkaline phosphatase returned to normal after nephrectomy but metastasis developed later without recurrent phosphatase elevation. CONCLUSIONS: Paraneoplastic serum alkaline phosphatase elevation in renal cell carcinoma patients implies an unfavorable prognosis, and additional paraneoplastic syndromes further worsen the prognosis. Recurrent or persistent serum alkaline phosphatase elevation after radical nephrectomy suggests distant metastasis or residual tumor. However, the return of serum alkaline phosphatase to normal does not guarantee cure of the disease. Identification of paraneoplastic serum alkaline phosphatase elevation is valuable in the prediction of outcome and postoperative followup of renal cell carcinoma patients.

Alkaline Phosphatase↗

PubMed1997

Clinical outcome at 3 months after transurethral vaporization of prostate for benign prostatic hyperplasia.

OBJECTIVES: To evaluate the clinical outcome of transurethral vaporization of the prostate (TUVP) for the management of benign prostatic hyperplasia (BPH). METHODS: Between March and June 1995, 30 patients with symptomatic BPH treated by TUVP were enrolled in this study. Transrectal ultrasonography (TRUS) was done preoperatively. American Urological Association (AUA) symptom score determination, pressure flow study, and questionnaire (for evaluating potency) were done preoperatively and 3 months postoperatively. RESULTS: The average age was 70.5 years (range 60 to 83) and estimated prostate size by TRUS before surgery was 33.8 +/- 14.0 g. The average AUA symptom score decreased significantly 3 months after TUVP (6.2 +/- 7.8 versus 18.2 +/- 9.0; P < 0.01). The maximum urine flow rate (Qmax) was 11.1 +/- 3.7 mL/min before TUVP (mean +/- SD) and 17.0 +/- 6.5 mL/min 3 months after TUVP, whereas the detrusor pressure at maximum urine flow (Pdes at Qmax) was 61.0 +/- 23.9 and 41.2 +/- 15.2 cm H2O, respectively. Qmax increased and Pdes at Qmax decreased significantly 3 months after TUVP. Of the 30 patients, 3 (10%) developed bladder neck contracture. Of the 24 patients who were potent sexually before operation, 3 (12.5%) developed impotence 3 months after surgery. CONCLUSIONS: TUVP is an effective alternative surgical procedure to relieve obstruction for patients with symptomatic BPH. However, cautious attitude on its usage is advocated based on our preliminary results indicating the occurrence of late complication such as impotence and bladder neck contracture.

Aged↗

PubMed1997

Probing calcium ion-induced conformational changes of Taiwan cobra phospholipase A2 by trinitrophenylation of lysine residues.

Phospholipase A2 (PLA2) from Naja naja atra (Taiwan cobra) snake venom was subjected to lysine modification with trinitrobenzene sulfonate (TNBS). Three major derivatives, TNP-1, TNP-2, and TNP-3, were separated by high-performance liquid chromatography (HPLC) from the reaction mixtures in the absence of Ca2+. However, only TNP-2 and TNP-3 were isolated when trinitrophenylated reaction was carried out in the presence of Ca2+. TNP-1 and TNP-2 contained only one TNP group, on Lys-65 and Lys-6, respectively; and both Lys-6 and Lys-65 were modified in TNP-3. The extent of modification on Lys-6 and Lys-65 was calculated from the peak areas of TNP proteins in the HPLC profile. It was found that the susceptibility of Lys-6 toward TNBS markedly increased by the addition of Ca2+ when Ca2+ concentration was higher than 5 mM. With regard to the involvement of Lys-6 in the binding of substrate, the increase in the reactivity of Lys-6 may arise from a conformational change around Lys-6 for binding with substrate in the presence of Ca2+. Alternatively, the nonessentiality of Lys-65 for PLA2 activity was revealed by the finding that TNP-1 still retained 95% activity of native enzyme. Moreover, the reactivity of Lys-65 toward TNBS did not greatly change in either the absence or presence of Ca2+, suggesting that Ca2+ binding did not cause an appreciable change in the microenvironment around Lys-65. These results indicate that the differential reactivities of Lys-6 and Lys-65 toward TNBS as affected by the binding of Ca2+ are well consistent with their functional roles in the catalytic mechanism of PLA2, and suggest that the occurrence of conformational changes with PLA2 could be explored by chemical modification studies.

Amino Acid Sequence↗

PubMed1997

The structural variations of epsilon-amino groups in phospholipase A2 enzymes from Naja naja atra and Bungarus multicinctus venoms.

Comparative studies on Naja naja atra phospholipase A2 (NNA-PLA2), Bungarus multicinctus phospholipase A2 (BM-PLA2), and their Lys-modified derivatives were made to assess the differences in the fine structures around the conserved Lys residues of PLA2 enzymes. It has found that the accessibility of Lys residues of PLA2 enzymes toward modified reagent, trinitrobenzene sulfonate, were not the same. Moreover, the extent of decrease in pI values of PLA2 enzymes that resulted from trinitrophenylation of lysine residues was different between NNA-PLA2 and BM-PLA2. The Lys-6 of BM-PLA2 mostly contributed to the positively charged character of the enzyme molecule, whereas the contribution of Lys-6 of NNA-PLA2 to its molecular charge was not notably different from other Lys residues. A linear relationship was observed by plotting the mobilities of PLA2 enzymes and their TNP derivatives against their pI values. However, native and Lys-modified NNA-PLA2 were not aligned with those of BM-PLA2 in the same line. Apparently the gross conformation of PLA2 enzymes was not notably perturbed by the modification of Lys residues, but the fine structure of NNA-PLA2 was not the same as that of BM-PLA2. These results indicate that the positioning of side chains of the conserved Lys residues in the two PLA2 enzymes is essentially different, and suggest that the variations in the fine structures of homologous proteins could be effectively explored by chemical modification studies and electrophoretic analysis.

Amino Acid Sequence↗