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D Cai

Publications and source records attributed to D Cai.

At least 91 records · Page 5Linked to original sources
PubMed1994

Copper amine oxidase: heterologous expression, purification, and characterization of an active enzyme in Saccharomyces cerevisiae.

A copper amine oxidase gene from a methylotrophic yeast Hansenula polymorpha has been expressed in Saccharomyces cerevisiae under the control of the ADHI promoter and the recombinant protein purified to near homogeneity. The recombinant enzyme is as active as the native enzyme in catalyzing methylamine oxidation. We demonstrate that it is a quinoprotein by redox-cycling staining and titrations with carbonyl reagents. The absorption spectral properties of the recombinant amine oxidase and its phenylhydrazine derivative are very similar to those of other copper amine oxidases. The cofactor in the enzyme is 2,4,5-trihydroxyphenylalanine (topa) quinone, as demonstrated by the pH-dependent shift in the lambda max of the p-nitrophenylhydrazone adduct. Alignment of an active-site peptide and DNA-derived protein sequences reveals a tyrosine residue as the precursor to topa quinone, consistent with findings with other copper amine oxidases. All evidence presented herein indicates that the heterologously expressed copper amine oxidase protein is processed posttranslationally in S. cerevisiae to form an active enzyme with an intact cofactor. This occurs despite an inability of S. cerevisiae to utilize amines as a nitrogen source. The implications of this study for the mechanism of topa quinone biogenesis are discussed.

Amine Oxidase (Copper-Containing)↗
PubMed1994

Primary structures for a mammalian cellular and serum copper amine oxidase.

The 6-hydroxydopa quinone-containing active site peptide from bovine serum amine oxidase has been found to be highly homologous to a segment of a cloned human kidney amiloride-binding protein (Barbry, P., Champe, M., Chassande, O., Munemitsu, S., Champigny, G., Lingueglia, E., Maes, P., Frelin, C., Tartar, A., Ullrich, A., and Lazdunski, M. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7347-7351). Additionally, a second 38-residue tryptic peptide (peptide XI) isolated from bovine serum amine oxidase shows 82% identity with a portion near the carboxyl terminus of the human kidney amiloride-binding protein. When an extended active site peptide was isolated from porcine kidney diamine oxidase (Janes, S. M., Palcic, M. M., Scaman, C. H., Smith, A. J., Brown, D. E., Dooley, D. M., Mure, M., and Klinman, J. P. (1992) Biochemistry 31, 12147-12154), it was found to be fully contained in the human kidney amiloride-binding protein. Examination of amiloride binding to bovine serum amine oxidase and porcine kidney diamine oxidase reveals dissociation constants of 196 and 9.1 microM, respectively. Taken together, these findings indicate that the cDNA isolated for human kidney amiloride-binding protein encodes a human kidney diamine oxidase. Two oligonucleotides, based on the tryptic peptide XI and active-site peptide of bovine serum amine oxidase, were used to amplify a portion of cDNA from a commercial bovine liver cDNA library through the use of the polymerase chain reaction. A full-length clone (2.7 kilobase pairs) for bovine serum amine oxidase was subsequently obtained through screening of the same cDNA library with the amplified 0.7-kilobase pair cDNA. These studies provide the first primary sequences for a mammalian cellular and serum copper amine oxidase. Computer alignment of amine oxidase cDNA-derived protein sequences reveals three conserved histidine residues, which are likely to be ligands to copper.

Amine Oxidase (Copper-Containing)↗
PubMed1994

The presequence of a chimeric construct dictates which of two mechanisms are utilized for translocation across the thylakoid membrane: evidence for the existence of two distinct translocation systems.

The translocation of plastocyanin across the thylakoid membrane in Pisum sativum has been studied in reconstitution assays and using chimeric constructs. The reconstitution assays demonstrate that plastocyanin translocation is absolutely dependent on the presence of a stromal factor(s) and nucleotide triphosphates (NTPs), whereas neither element is required for the translocation of the 23 or 16 kDa proteins of the oxygen-evolving complex. Previous studies had revealed that the transthylakoidal delta pH is essential for translocation of the 23 and 16 kDa proteins but unnecessary for plastocyanin translocation. The basis for these mechanistic differences has been tested by analysing the translocation of a chimeric construct consisting of the presequence of the 23 kDa protein linked to the mature plastocyanin sequence. This construct is efficiently imported into thylakoids in the absence of stromal extracts or NTPs and translocation across the thylakoid membrane within intact chloroplasts is totally inhibited by the uncoupler nigericin: the translocation requirements are thus identical to those of the pre-23 kDa protein and diametrically opposite to those of pre-plastocyanin. Transport across the thylakoid membrane of a second fusion protein, consisting of the presequence of the 16 kDa protein linked to mature plastocyanin, is also dependent on a delta pH. The data suggest that two distinct systems are involved in the translocation of proteins across the thylakoid membrane, with each system recognizing specific signals within the presequences of a subset of lumenal protein precursors.

Amino Acid Sequence↗
PubMed1994

Effects of gap junction conductance on dynamics of sinoatrial node cells: two-cell and large-scale network models.

A computational model of single rabbit sinoatrial (SA) node cells has been revised to fit data on regional variation of rabbit SA node cell oscillation properties. The revised model simulates differences in oscillation frequency, maximum diastolic potential, overshoot potential, and peak upstroke velocity observed in cells from different regions of the node. Dynamic properties of electrically coupled cells, each with different intrinsic oscillation frequency, are studied as a function of coupling conductance. Simulation results demonstrate at least four distinct regimes of behavior as coupling conductance is varied: a) independent oscillation (Gc < 1 pS); b) complex oscillation (1 < or = Gc < 220 pS); c) frequency, but not waveform entrainment (Gc > or = 220 pS); and d) frequency and waveform entrainment (Gc > or = 50 nS). The conductance of single cardiac myocyte gap junction channels is about 50 pS. These simulations therefore show that very few gap junction channels between each cell are required for frequency entrainment. Analyses of large-scale SA node network models implemented on the Connection Machine CM-200 supercomputer indicate that frequency entrainment of large networks is also supported by a small number of gap junction channels between neighboring cells.

Algorithms↗
PubMed1993

Lignin-degrading peroxidases of Phanerochaete chrysosporium.

Lignin and manganese peroxidases are secreted by the basidiomycete Phanerochaete chrysosporium during secondary metabolism. These enzymes play major roles in lignin degradation. The active site amino acid sequence of these lignin-degrading peroxidases is similar to that of horseradish peroxidase (HRP) and cytochrome c peroxidase (CcP). The mechanism by which they oxidize substrates also appears to be the similar. pH has a similar effect on lignin peroxidase compound I formation as on HRP or CcP; however, the pKa controlling compound I formation for lignin peroxidase appears to be much lower. Lignin-degrading peroxidases are able to catalyze the oxidation of substrates with high redox potential. This unique ability is consistent with a heme active site of low electron density, which is indicated by high redox potential.

Amino Acid Sequence↗
PubMed1993

Doppler flowmeter-assessed circadian rhythms in neonatal cardiac function, family history, and intrauterine growth retardation.

This study examines the circadian variation of aortic blood flow velocity, acceleration, and distance of stroke, assessed every 4 hours for 24 hours by Doppler ultrasound in 88 human newborns. Groups 1 and 2 involve neonates with a negative family history of high blood pressure or myocardial infarction monitored during the first day or between the second and tenth days postpartum, respectively; groups 3 to 6 are 2- to 10-day-old babies with a positive family history of high blood pressure (group 3) or of myocardial infarction (group 4) or with a negative family history and intrauterine growth retardation (group 5) or born prematurely (group 6). Cosinor analyses demonstrate a circadian rhythm for all variables in groups 2 and 5 (p < 0.001), with overall high values occurring around 17:40. In group 2, a circadian rhythm remains demonstrable in boys and girls considered separately, and mostly so in arbitrary subgroups of different sizes, with only slight differences in rhythm parameters among them. These results show that, with relatively small samples, reproducible circadian rhythms of cardiac function can be detected shortly after birth in neonates with a negative family history of high blood pressure or myocardial infarction.

Analysis of Variance↗
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

Study of the effect of lithium on lymphokine-activated killer cell activity and its antitumor growth.

The in vitro effect of lithium on lymphokine-activated killer cell (LAK) activity and its in vivo antitumor growth were observed. LAK activity was enhanced when LiCl was added during LAK cell induction, and this enhancement was observed both in human peripheral blood mononuclear cell and in mouse splenocytes used as LAK precursors. Cholera toxin, which can increase intracellular levels of cAMP, decreased LAK cell activity. However, lithium partially reversed this inhibitory effect, indicating that lithium increased LAK cell activity by decreasing cAMP levels. D-Sphingosine, an inhibitor of protein kinase C, and EGTA, a calcium chelator, both inhibited the LAK cell activity. However, their inhibitory effects could not be reversed by lithium because lithium was added in the culture in combination with one of these inhibitors during LAK cell induction. By using slot blot analysis, the effect of lithium on the expression of tumor necrosis factor-alpha mRNA of LAK cells was analyzed. Lithium increased the level of tumor necrosis factor-alpha mRNA when both lithium and interleukin 2 were added to induce LAK cells. The in vivo antitumor effect of lithium has also been studied. Using a mouse melanoma experimental model, the effect of lithium on tumor growth was also observed. Both lithium alone and interleukin 2/LAK had an antitumor effect, whereas the treatment of interleukin 2/LAK in combination with lithium had the strongest inhibitory effect on tumor growth, since this treatment resulted in reduction of tumor size and prolongation of survival in tumor-bearing mice. Therefore, it is hopeful that lithium can be used as a new immunomodulator for cancer immunotherapy and immune diseases.

Animals↗