Biomedical subjects

M Tien

Publications and source records attributed to M Tien.

At least 55 records · Page 3Linked to original sources

PubMed1992

Production and characterization of recombinant lignin peroxidase isozyme H2 from Phanerochaete chrysosporium using recombinant baculovirus.

Recombinant Phanerochaete chrysosporium lignin peroxidase isozyme H2 (pI 4.4) was produced in insect cells infected with a genetically engineered baculovirus containing a copy of the cDNA clone lambda ML-6. The recombinant enzyme was purified to near homogeneity and is capable of oxidizing veratryl alcohol, iodide, and, to a lesser extent, guaiacol. The Km of the recombinant enzyme for veratryl alcohol and H2O2 is similar to that of the fungal enzyme. The guaiacol oxidation activity or any other activity is not dependent upon Mn2+. The purified recombinant peroxidase is glycosylated with N-linked carbohydrate(s). The recombinant lignin peroxidase eluted from an anion exchange resin similar to that of native isozyme H1 rather than H2. However, the pI of the recombinant enzymes is different from both H1 and H2 isozymes. Further characterization of native isozymes H1 and H2 from the fungal cultures revealed identical N-terminus residues. This indicates that isozymes H1 and H2 differ in post-translational modification.

Amino Acid Sequence↗

PubMed1992

Kinetic studies on the formation and decomposition of compounds II and III. Reactions of lignin peroxidase with H2O2.

The present study characterizes the serial reactions of H2O2 with compounds I and II of lignin peroxidase isozyme H1. These two reactions constitute part of the pathway leading to formation of the oxy complex (compound III) from the ferric enzyme. Compounds II and III are the only complexes observed; no compound III* is observed. Compound III* is proposed to be an adduct of compound III with H2O2, formed from the complexation of compound III with H2O2 (Wariishi, H., and Gold, M. H. (1990) J. Biol. Chem. 265, 2070-2077). We provide evidence that demonstrates that the spectral data, on which the formation of compound III* is based, are merely an artifact caused by enzyme instability and, therefore, rule out the existence of compound III*. The reactions of compounds II and III with H2O2 are pH-dependent, similar to that observed for reactions of compounds I and II with the reducing substrate veratryl alcohol. The spontaneous decay of the compound III of lignin peroxidase results in the reduction of ferric cytochrome c. The reduction is inhibited by superoxide dismutase, indicating that superoxide is released during the decay. Therefore, the lignin peroxidase compound III decays to the ferric enzyme through the dissociation of superoxide. This mechanism is identical with that observed with oxymyoglobin and oxyhemoglobin but different from that for horseradish peroxidase. Compound III is capable of reacting with small molecules, such as tetranitromethane (a superoxide scavenger) and fluoride (a ligand for the ferric enzyme), resulting in ferric enzyme and fluoride complex formation, respectively.

Anisoles↗

PubMed1992

Heterogeneity and regulation of manganese peroxidases from Phanerochaete chrysosporium.

Lignin and Mn peroxidases are two families of isozymes produced by the lignin-degrading fungus Phanerochaete chrysosporium under nutrient nitrogen or carbon limitation. We purified to homogeneity the three major Mn peroxidase isozymes, H3 (pI = 4.9), H4 (pI = 4.5), and H5 (pI = 4.2). Amino-terminal sequencing of these isozymes demonstrates that they are encoded by different genes. We also analyzed the regulation of these isozymes in carbon- and nitrogen-limited cultures and found not only that the lignin and Mn peroxidases are differentially regulated but also that differential regulation occurs within the Mn peroxidase isozyme family. The isozyme profile and the time at which each isozyme appears in secondary metabolism differ in both nitrogen- and carbon-limited cultures. Each isozyme also responded differently to the addition of a putative inducer, divalent Mn. The stability of the Mn peroxidases in carbon- and nitrogen-limited cultures was also characterized after cycloheximide addition. The Mn peroxidases are more stable in carbon-limited cultures than in nitrogen-limited cultures. They are also more stable than the lignin peroxidases. These data collectively suggest that the Mn peroxidase isozymes serve different functions in lignin biodegradation.

Amino Acid Sequence↗

PubMed1991

Heterologous expression of active manganese peroxidase from Phanerochaete chrysosporium using the baculovirus expression system.

The cDNA encoding Mn peroxidase isozyme H4 from Phanerochaete chrysosporium was recombined into a baculovirus and heterologously expressed in Sf9 cells. The recombinant Mn peroxidase has the same molecular weight as the native enzyme as determined by SDS-PAGE and cross-reacts with a Mn peroxidase-specific antibody. The recombinant enzyme has a slightly lower pI than the native fungal isozyme H4 indicating some differences in post-translational modification. Phenol red, guaiacol, and vanillylacetone, substrates of the native Mn peroxidase, are oxidized by the recombinant enzyme. All of the activities are dependent on both Mn (II) and H2O2.

Baculoviridae↗

PubMed1991

Proton NMR investigation into the basis for the relatively high redox potential of lignin peroxidase.

Lignin peroxidase shares several structural features with the well-studied horseradish peroxidase and cytochrome c peroxidase but carries a higher redox potential. Here the heme domain of lignin peroxidase and the lignin peroxidase cyanide adduct was examined by 1HNMR spectroscopy, including nuclear Overhauser effect and two-dimensional measurements, and the findings were compared with those for horseradish peroxidase and cytochrome c peroxidase. Structural information was obtained on the orientation of the heme vinyl and propionate groups and the proximal and distal histidines. The shifts of the epsilon1 proton of the proximal histidine were found to be empirically related to the Fe3+/Fe2+ redox potentials.

Journal Article↗

PubMed1991

Lignin peroxidase of Phanerochaete chrysosporium. Evidence for an acidic ionization controlling activity.

The active site amino acid residues of lignin peroxidase are homologous to those of other peroxidases; however, in contrast to other peroxidases, no pH dependence is observed for the reaction of ferric lignin peroxidase with H2O2 to form compound I (Andrawis, A., Johnson, K.A., and Tien, M. (1988) J. Biol. Chem. 263, 1195-1198). Chloride binding is used in the present study to investigate this reaction further. Chloride binds to lignin peroxidase at the same site as cyanide and hydrogen peroxide. This is indicated by the following. 1) Chloride competes with cyanide in binding to lignin peroxidase. 2) Chloride is a competitive inhibitor of lignin peroxidase with respect to H2O2. The inhibition constant (Ki) is equal to the dissociation constant (Kd) of chloride at all pH values studied. Chloride binding is pH dependent: chloride binds only to the protonated form of lignin peroxidase. Transient-state kinetic studies demonstrate that chloride inhibits lignin peroxidase compound I formation in a pH-dependent manner with maximum inhibition at low pH. An apparent pKa was calculated at each chloride concentration; the pKa increased as the chloride concentration increased. Extrapolation to zero chloride concentration allowed us to estimate the intrinsic pKa for the ionization in the lignin peroxidase active site. The results reported here provide evidence that an acidic ionizable group (pKa approximately 1) at the active site controls both lignin peroxidase compound I formation and chloride binding. We propose that the mechanism for lignin peroxidase compound I formation is similar to that of other peroxidases in that it requires the deprotonated form of an ionizable group near the active site.

Benzyl Alcohols↗

PubMed1991

Overproduction of lignin-degrading enzymes by an isolate of Phanerochaete chrysosporium.

Phanerochaete chrysosporium is a white rot fungus which secretes a family of lignin-degrading enzymes under nutrient limitation. PSBL-1 is a mutant of this organism that generates the ligninolytic system under nonlimiting conditions during primary metabolism. Lignin peroxidase, manganese peroxidase, and glyoxal oxidase activities for PSBL-1 under nonlimiting conditions were 4- to 10-fold higher than those of the wild type (WT) under nitrogen-limiting conditions. PSBL-1 was still in the log phase of growth while secreting the enzymes, whereas the WT had ceased to grow by this time. As in the WT, manganese(II) increased manganese peroxidase activity in the mutant. However, manganese also caused an increase in lignin peroxidase and glyoxal oxidase activities in PSBL-1. Addition of veratryl alcohol to the culture medium stimulated lignin peroxidase activity, inhibited glyoxal oxidase activity, and had little effect on manganese peroxidase activity in PSBL-1, as in the WT. Fast protein liquid chromatography (FPLC) analysis shows production of larger amounts of isozyme H2 in PSBL-1 than in the WT. These properties make PSBL-1 very useful for isolation of large amounts of all ligninolytic enzymes for biochemical study, and they open the possibility of scale-up production for pratical use.

Alcohol Oxidoreductases↗

PubMed1990

Characterization of the oxycomplex of lignin peroxidases from Phanerochaete chrysosporium: equilibrium and kinetics studies.

The oxycomplexes (compound III, oxyperoxidase) of two lignin peroxidase isozymes, H1 (pI = 4.7) and H8 (pI = 3.5), were characterized in the present study. After generation of the ferroperoxidase by photochemical reduction with deazoflavin in the presence of EDTA, the oxycomplex is formed by mixing ferroperoxidase with O2. The oxycomplex of isozyme H8 is very stable, with an autoxidation rate at 25 degrees C too slow to measure at pH 3.5 or 7.0. In contrast, the oxycomplex of isozyme H1 has a half-life of 52 min at pH 4.5 and 29 min at pH 7.5 at 25 degrees C. The decay of isozyme H1 oxycomplex follows a single exponential. The half-lives of lignin peroxidase oxycomplexes are much longer than those observed with other peroxidases. The binding of O2 to ferroperoxidase to form the oxycomplex was studied by stopped-flow methods. At 20 degrees C, the second-order rate constants for O2 binding are 2.3 X 10(5) and 8.9 X 10(5) M-1 s-1 for isozyme H1 and 6.2 X 10(4) and 3.5 X 10(5) M-1 s-1 for isozyme H8 at pH 3.6 and pH 6.8, respectively. The dissociation rate constants for the oxycomplex of isozyme H1 (3.8 Z 10(-3) s-1) and isozyme H8 (1.0 X 10(-3) s-1) were measured at pH 3.6 by CO trapping. Thus, the equilibrium constants (K, calculated from kon/koff) for both isozymes H1 (7.0 X 10(7) M-1) and H8 (6.2 X 10(7) M-1) are higher than that of myoglobin (1.9 Z 10(6) M-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Benzyl Alcohols↗

PubMed1990

Chronological changes in genetic variance and heritability of anthropometric characteristics among Chinese twin infants.

In order to examine the chronologic changes in genetic variance and heritability of anthropometric characteristics of Chinese infants in Taiwan, a total of 521 pairs of same-sexed twin neonates given birth in four major general teaching hospitals in Taipei City were studied. Based on the placental pattern and 12 red blood cell antigens, 428 MZ and 93 DZ twin pairs were identified and followed up to the age of one year. There was no significant genetic variance for all anthropometric characteristics adjusted for sex and gestational week before the age of six months. After adjusting for sex and gestational week, a significant genetic variance was observed at the age of six months, with heritability values of 0.51 (weight), 0.63 (head circumference), 0.77 (chest circumference), and 0.53 (arm circumference), as well as at one year, although with considerably lower heritability values. This implies that growth is dynamically determined by both genetic and environmental factors during infancy.

Age Factors↗

PubMed1990

Genetic variance and heritability of temperament among Chinese twin infants.

In order to examine the genetic variance and heritability of temperament among Chinese infants in Taiwan, a total of 62 pairs of same-sexed twin infants given birth in four major general teaching hospitals in Taipei City were studied. Based on placentation and 12 red blood cell antigens, 44 MZ and 18 DZ pairs were identified. Temperament was assessed at the age of six months by the Chinese edition of Carey's Temperament scale. Significant genetic variance was observed for activity level, approach or withdrawal, intensity of reaction, quality of mood, and threshold of responsiveness, with a heritability of 0.64, 0.56, 0.74, 0.39, and 0.45, respectively. There was no significant intrapair difference in temperamental characteristics between monochorionic and dichorionic MZ twins.

Female↗

PubMed1990

Chronological changes in genetic variance and heritability of systolic and diastolic blood pressure among Chinese twin neonates.

In order to examine the chronological changes in genetic variance and heritability of arterial systolic and diastolic blood pressure (SBP and DBP of Chinese infants in Taiwan, a total of 339 same-sexed twin neonates born in four major general teaching hospitals in Taipei City were studied. Based on placentation and 12 red blood cell antigens, 274 monozygotic (MZ) and 65 dizygotic (DZ) twin pairs were identified and followed up to the age of one year. Both SBP and DBP were measured by Doppler blood pressure monitor. Within-pair mean squares of SBP and DBP were consistently smaller in MZ than DZ twins at ages one month and over. The findings remained unchanged after the adjustment for the effects of age, sex, gestational age, placentation and physical state during blood pressure measurement. Falconer's heritability indices for adjusted SBP and DBP at ages two months and over ranged from 0.29 to 0.55 and from 0.27 to 0.45, respectively. The study indicates an important genetic influence on blood pressure during infancy.

Blood Pressure↗

PubMed1990

Genetic variance and heritability of serum cholesterol and triglycerides among Chinese twin neonates.

In order to examine the genetic variance and heritability of serum total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglycerides, a total of 349 pairs of same-sexed twin neonates born in four major general teaching hospitals in Taipei City were studied. Based on the placental pattern and 12 red blood cell antigens, 271 monozygotic (MZ) and 78 dizygotic (DZ) twin pairs were identified. There was a significant genetic variance for total cholesterol, HDL-C, LDL-C, and triglycerides both unadjusted and adjusted for sex, gestational age and placentation. The unadjusted heritability of total cholesterol, HDL-C, LDL-C, and triglycerides was 0.59, 0.30, 0.25 and 0.75, respectively; while the corresponding adjusted heritability was 0.74, 0.38, 0.31, and 0.49, respectively. Intrapair variance of serum lipids was not significantly different between monochorionic and dichorionic MZ twins.

China↗

PubMed1990

Selection and characterization of mutants of Phanerochaete chrysosporium exhibiting ligninolytic activity under nutrient-rich conditions.

Synthesis of the ligninolytic system of the wood-degrading fungus Phanerochaete chrysosporium is induced during secondary metabolism, brought about by nitrogen, carbon, or sulfur starvation. We describe here a strategy for selection of mutants which are ligninolytic (lignin----CO2) and overproduce lignin-degrading enzymes (ligninases) under nutrient-rich conditions (during primary metabolism). The strategy is based on using an adduct of lysine and a lignin model compound. Ligninase-dependent oxidation of this adduct releases free lysine, which complements the lysine requirements of a lysine auxotroph. Accordingly, a lysine auxotroph was mutagenized by UV irradiation and survivors were plated onto medium containing the adduct and high ammonia nitrogen. Four mutants which overproduce the ligninase isozymes were isolated by this procedure. Further characterization of one of the mutants, PSBL-1, indicated that the predominant isozymes produced are H1 (pI = 4.7) and H2 (pI = 4.4). The ligninase activity of PSBL-1, measured by veratryl alcohol oxidation, peaks on day 5 at over 1,000 U.liter-1. The mutant PSBL-1 was also able to degrade [14C]lignin to 14CO2, indicating that the complete ligninolytic system is deregulated.

Basidiomycota↗

PubMed1989

Phosphorylation of lignin peroxidases from Phanerochaete chrysosporium. Identification of mannose 6-phosphate.

Many of the extracellular lignin-degrading peroxidases from the wood-degrading fungus Phanerochaete chrysosporium are phosphorylated. Immunoprecipitation of the extracellular fluid of cultures grown with H2K32PO4 with a polyclonal antibody raised against one of the lignin peroxidase isozymes, H8 (pI 3.5), revealed the incorporation of H2K32PO4 into lignin peroxidases. Analyses of the purified isozymes from labeled cultures by isoelectric focusing showed that, in addition to isozyme H8, lignin peroxidase isozymes H2 (pI 4.4), H6 (pI 3.7), and H10 (pI 3.3) are also phosphorylated. These analyses also showed that lignin peroxidase isozyme H1 (pI 4.7) and manganese-dependent peroxidase isozymes H3 (pI 4.9) and H4 (pI 4.5) are not phosphorylated. Phosphate quantitation indicated the presence of one molecule of phosphate/molecule of enzyme for all of the phosphorylated isozymes. To locate the site of phosphorylation, one-dimensional phosphoamino acid analysis was performed with hydrolyzed 32P-protein. However, phosphotyrosine, phosphoserine, and phosphothreonine could not be identified. Coupled enzyme assays of acid hydrolysate indicated the presence of mannose 6-phosphate as the phosphorylated component on the lignin peroxidase isozymes. Digestion of the isozymes with N-glycanase released the phosphate component, indicating that the mannose 6-phosphate is contained on an asparagine-linked oligosaccharide.

Agaricales↗

PubMed1989

Oxidation-reduction potentials and ionization states of extracellular peroxidases from the lignin-degrading fungus Phanerochaete chrysosporium.

The oxidation-reduction potentials of lignin peroxidase isozymes H1, H2, H8, and H10 as well as the Mn-dependent peroxidase isozymes H3 and H4 are reported. The potentiometric titrations involving the ferrous and ferric states of the enzyme had Nernst plots indicating single-electron transfer. The Em7 values of lignin peroxidase isozymes H1, H2, H8, and H10 are -142, -135, -137, and -127 mV versus standard hydrogen electrode, respectively. The Em7 values for the Mn-dependent peroxidase isozymes H3 and H4 are -88 and -93 mV versus standard hydrogen electrode, respectively. The midpoint potential of H1, H8, and H4 remained unchanged in the presence of their respective substrates, veratryl alcohol and Mn(II). The midpoint potential between the ferric and ferrous forms of isozymes H1 and H4 exhibited a pH-dependent change between pH 3.5 and pH 6.5. These results indicate that the reductive half-reaction of the enzymes is the following: ferric peroxidase + le- + H+----ferrous peroxidase. Above pH 6.5, the effect of pH on the midpoint potential is diminished and indicates that an ionization with an apparent pKa equal to approximately 6.6-6.7 occurs in the reduced form of the enzymes. A heme-linked ionization group in the ferrous form of the enzymes was confirmed by studying the effect of pH on the absorption spectra of isozymes H1 and H4. These spectrophotometric pH titration experiments confirmed the electrochemical results indicating pKa values of 6.59 and 6.69 for reduced isozymes H1 and H4, respectively. These results indicate the presence of a heme-linked ionization of an amino acid in the reduced form of the lignin peroxidase isozymes similar to that of other plant peroxidases.

Basidiomycota↗

PubMed1989

Manganese-dependent peroxidase from Phanerochaete chrysosporium. Primary structure deduced from cDNA sequence.

A cDNA clone encoding a manganese-dependent peroxidase from the filamentous fungus Phanerochaete chrysosporium was isolated and characterized. The clone, lambda MP-1, was isolated by screening a lambda gt11 expression library with polyclonal antibodies raised against a purified manganese-dependent peroxidase (isozyme H4, pI 4.5). The lambda MP-1 cDNA sequence predicts a mature protein containing 358 amino acids with a molecular weight of 37,711 preceded by a leader peptide of 24 amino acid residues. The N-terminal amino acid sequence of a purified manganese-dependent peroxidase (H4) corresponds to the sequence deduced from the cDNA. Some homology (58% in nucleotide sequence and 65% in amino acid sequence) is observed between the manganese-dependent peroxidase and lignin peroxidase isozyme H8. The highest degree of similarity is observed near the enzyme active site. Residues essential for peroxidase activity, the distal and proximal histidines, can be identified in the amino acid sequence. Near these residues, homology is also observed with several other peroxidases. Northern blot analysis of poly(A)+ RNA from nitrogen-limited P. chrysosporium cultures indicates that the level of messenger RNA correlates with expression of the enzyme and its activity. This is consistent with the regulation of the enzyme being at the level of transcription.

Agaricales↗

PubMed1989

Characterization of two lignin peroxidase clones from Phanerochaete chrysosporium.

Two cDNA clones encoding lignin peroxidase isozymes from Phanerochaete chrysosporium have been isolated and characterized. One of the clones, lambda ML-4, encodes isozyme H8 as does the previously reported clone lambda ML-1 [Tien, M. and Tu, C.-P.D. Nature 326 (1987) 520-523; 328, 742]. Our data are consistent with lambda ML-1 and lambda ML-4 being allelic variants. The other clone, lambda ML-5, encodes a homologous isozyme. We have also isolated the genomic clone corresponding to lambda ML-4 cDNA. Conserved residues thought to be essential for peroxidase function were identified in the predicted amino acid sequences of both cDNA clones. Northern blot analyses indicate that these isozymes are expressed during secondary metabolism, appearing on day 4 of growth and increasing on days 5 and 6.

Amino Acid Sequence↗

PubMed1989

On the reactions of lignin peroxidase compound III (isozyme H8).

Compound III (oxyperoxidase) of lignin peroxidase isozyme H8 (pI = 3.5) is formed by either reduction of native ferric enzyme (to ferrous) followed by the reaction with dioxygen or by the addition of excess hydrogen peroxide to resting enzyme. When prepared from the ferrous enzyme, Compound III is stable for days. When formed from excess hydrogen peroxide, the enzyme is rapidly inactivated. However, if the hydrogen peroxide is removed by gel filtration, the resulting Compound III exhibits the same stability as when prepared from ferrous enzyme. Compound III of lignin peroxidase is also relatively unreactive to reducing substrates. Addition of veratryl alcohol to Compound III does not result in any reaction. However, when only 1 equivalent of hydrogen peroxide is added to Compound III in the presence of veratryl alcohol, Compound III is converted to resting enzyme and veratraldehyde formation is detected spectroscopically.

Benzyl Alcohols↗