Biomedical subjects

S H Lin

Publications and source records attributed to S H Lin.

At least 217 records · Page 12Linked to original sources

PubMed1989

Cloning and expression of a cDNA coding for a rat liver plasma membrane ecto-ATPase. The primary structure of the ecto-ATPase is similar to that of the human biliary glycoprotein I.

The amino acid sequence of the ecto-ATPase from rat liver was deduced from analysis of cDNA clones and a genomic clone. Immunoblots with antibodies raised against a peptide sequence deduced from the cDNA sequence indicated that the determined amino acid sequence is that of the ecto-ATPase. The deduced sequence predicts a 519-amino acid protein with a calculated molecular mass of 57,388 daltons. There are 16 potential asparagine-linked glycosylation sites in the protein. Hydropathy analysis of the deduced amino acid sequence indicates that the protein has two hydrophobic stretches. One is located at the N-terminal and the other is near the C-terminal end. A full-length clone encoding the ecto-ATPase was expressed transiently in mouse L cells and human HeLa cells. The cell lysate from the transfected cells contained immunoreactive ecto-ATPase and Ca2+-stimulated ATPase activities. The expressed protein is glycosylated and has an apparent molecular weight (100,000) similar to that of the rat liver plasma membrane ecto-ATPase.

Amino Acid Sequence↗

PubMed1989

Resonance Raman assignment and evidence for noncoupling of individual 2- and 4-vinyl vibrational modes in a monomeric cyanomethemoglobin.

We have investigated the resonance Raman spectra of monomeric insect cyanomethemoglobins (CTT III and CTT IV) reconstituted with (1) protohemes IX selectively deuterated at the 4-vinyl as well as the 2,4-divinyls, (2) monovinyl-truncated hemes such as pemptoheme (2-hydrogen, 4-vinyl) and isopemptoheme (2-vinyl, 4-hydrogen), (3) symmetric hemes such as protoheme III (with 2- and 3-vinyls) and protoheme XIII (with 1- and 4-vinyls), and (4) hemes without 2- and 4-vinyls such as mesoheme IX, deuteroheme IX, 2,4-dimethyldeuteroheme IX, and 2,4-dibromodeuteroheme IX. Evidence is presented that the highly localized vinyl C = C stretching vibrations at the 2- and 4-positions of the heme in these cyanomet CTT hemoglobins are noncoupled and inequivalent; i.e., the 1631- and 1624-cm-1 lines have been assigned to 2-vinyl and 4-vinyl, respectively. The elimination of the 2-vinyl (in pemptoheme) or the 4-vinyl (in isopemptoheme) does not affect the C = C stretching frequency of the remaining vinyl. Furthermore, two low-frequency vinyl bending modes at 412 and 591 cm-1 exhibit greatly different resonance Raman intensities between 2-vinyl and 4-vinyl. The observed intensity at 412 cm-1 is primarily derived from 4-vinyl, whereas the 591-cm-1 line results exclusively from the 2-vinyl. Again, there is no significant coupling between 2-vinyl and 4-vinyl for these two bending modes.

Animals↗

PubMed1989

Characterization of the ethenoadenosine diphosphate binding site of myosin subfragment 1. Energetics of the equilibrium between two states of nucleotide.S1 and vanadate-induced global conformation changes detected by energy transfer.

The fluorescence decay of 1,N6-ethenoadenosine diphosphate (epsilon ADP) bound to myosin subfragment 1 (S1) was studied as a function of temperature. The decay was biexponential, and the two lifetimes were quenched relative to the single lifetime of free epsilon ADP. The temperature dependence of the fractional intensities of the decay components showed two states of the S1.epsilon ADP complex. At pH 7.5 in 30 mM TES, 60 mM KCl, and 3 mM MgCl2, the equilibrium constant for the conversion of the low-temperature state (S1L.epsilon ADP) to the high-temperature state (S1H.epsilon ADP) was 40 at physiological temperatures, and delta H degrees = 13 kcal.mol-1 and delta S degrees = 49 cal.deg-1.mol-1. At 10 degrees C the equilibrium constant of S1 for epsilon ADP was 5, indicating that S1H.epsilon ADP was the dominant state, and that for the vanadate complex epsilon ADP.Vi was 0.7, suggesting that in S1.epsilon ADP.Vi the dominant state of the S1-nucleotide complex was converted from S1H.epsilon ADP to S1L.epsilon ADP. The single rotational correlation time of bound epsilon ADP at 10 degrees C decreased from 107 ns in S1.epsilon ADP to 74 ns in S1+.epsilon ADP.Vi. Conversion of the binary complex to the ternary vanadate complex resulted in a 3-A decrease in the energy transfer distance between bound epsilon ADP and N-[4-(dimethylamino)-3,5-dinitrophenyl]maleimide attached to SH1 and a decrease of the average distance between bound epsilon ADP and bound Co2+ from 12.6 to 8.3 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1989

Resonance Raman enhancement of the Mn-N-O bending mode in nitrosyl manganese "strapped" and "open" heme complexes.

Resonance Raman spectra of the MnII-NO moiety in synthetic nitrosyl manganese heme complexes with and without steric hindrance are reported. The "strapped" hemes having a hydrocarbon strap (variable length) across one face of the heme hinder the perpendicular bonding of a linear ligand. These complexes were employed to investigate the effects of ligand distortion (primarily tilting) on Mn-NO stretching, Mn-N-O bending, and N-O stretching modes. It is demonstrated that ligand distortion in the MnII-NO system is a valid mechanism for causing the resonance enhancement of the Mn-N-O bending mode, similar to that observed in the FeII-CO system (Yu, N.-T., E. A. Kerr, B. Ward, and C. K. Chang. 1983. Biochemistry. 22:4534-4540). More interesting is the observation of the delta(Mn-N-O) enhancement caused by the tilting of the trans Mn-N epsilon bond in the "open" heme complexes (e.g., heme-5 and proto-1X dimethylester) with 1,2-dimethylimidazole or piperidine as a base. The nu(Mn-NO) and nu(N-O) modes exhibit an increase and a decrease, respectively, as the strap length decreases (hence the steric hindrance increases). Both nu(Mn-NO) and nu(N-O) frequencies are insensitive to the strength of the trans base. The results from "strapped" and "open" model heme systems imply that the Mn-N-O geometry is essentially linear and perpendicular in the nitrosyl complexes of monomeric manganese insect hemoglobin CTT IV and sperm whale myoglobin. The unusually low nu(N-O) frequency in the manganese myoglobin complex may be caused by the distal histidine-NO interaction. The delta(Mn-N-O) enhancement in both nitrosyl manganese CTT IV and nitrosyl manganese myoglobin may be caused by a tilting of the Mn"-Nf (proximal histidine) bond.

Heme↗

PubMed1989

Polymorphic reticulosis: a malignant lymphoma of B-cell lineage.

Whether the pathogenesis of polymorphic reticulosis is from T cells, B cells, or histiocytes has been controversial. In this study, the Southern blot hybridization technique was used to analyze immunoglobulin and T-cell receptor beta-chain genes and to perform the conventional surface marker analysis in two patients with polymorphic reticulosis. The immunophenotype demonstrated the presence of predominantly mature, activated T-lymphocytes, minimal B-cells, and no natural killer cells or monocytes/granulocytes. The mature T-cell phenotype could be due to either inflammatory infiltrates or neoplastic cells of peripheral T-cell type, because the two coexist in polymorphic reticulosis tumors. The value of surface marker examination is limited in the analysis of PMR tumors. However, genetic analysis revealed that only Ig genes were rearranged, with no rearrangement of the TCR beta gene. Rearrangement of immunoglobulin genes occurs in B-lineage lymphoid neoplasms and is thought to be a criterion for diagnosis of lymphoid neoplasms. Based on genetic analysis and clinicopathologic information, this study concluded that polymorphic reticulosis is a malignant lymphoma of B-cell lineage.

Adult↗

PubMed1989

[Screening of antibodies to human T-cell leukemia virus type I (HTLV-I) in sera of blood donors and comparison of different methods].

Nine hundred and sixty five serum samples of blood donors from the blood bank of Veterans General Hospital Taipei were screened for antibodies to human T-cell leukemia virus type I (HTLV-I) by different methods. Adult T-cell Leukemia cell lines MT-1, MT-2, KH-2 and MT-2 stimulated by phorbol 12-myristate 13-acetate (PMA) were used as target cells in immunofluorescence microscopy (IF) examination with positive rates as 0.20% (2/965), 0.62% (6/965), 0.20% (2/965) and 0.51 (5/965) respectively. In addition to these, enzyme-linked immunosorbent assay (ELISA) gave a 2.59% (25/965) positive rate and particle agglutination (PA) test revealed a positive rate of 0.93% (9/965). The samples positive in IF were also positive in PA and those positive in PA were found positive in ELISA without exceptions. The 25 ELISA positive samples as well as 75 samples randomly chosen from the ELISA negative ones received Western blot (WB) analysis as a confirmation test. Only 3 of these 100 were considered positive in WB, giving a final positive rate of 0.31%, (3/965), and all of them were originally positive in both PA and ELISA. Particle agglutination test as the screening test and Western blot analysis as the confirming one were suggested to be a routine procedure for detecting anti-HTLV-I antibodies in blood banks through detailed comparison and discussion.

Adult↗

PubMed1989

Intrauterine single fetal demise in a twin gestation. A case report.

Intrauterine demise of one fetus in a twin gestation as a rare occurrence not only entails the higher premature and neonatal death rates, fetal distress or abnormal presentation on the surviving twin, but also threatens the mother with toxemia. The consumptive coagulopathy imposes a potential risk of health on both mother and the surviving fetus or neonate. We herein report a 29-year-old nullipara in such case who finally delivered a stillborn and a living one. High risk management and careful pediatric follow-up are recommended.

Adult↗

PubMed1988

Membrane-buffer partition coefficients of tetracaine for liquid-crystal and solid-gel membranes estimated by direct ultraviolet spectrophotometry.

The membrane-buffer partition coefficient of tetracaine was measured by direct ultraviolet spectrophotometry in dimyristoylphosphatidylcholine unilamellar liposomes at temperatures above and below the main phase transition. The partition coefficients of uncharged tetracaine to solid-gel (18 degrees C) and liquid-crystal (30 degrees C) membranes were 6.9 x 10(4) and 1.2 x 10(5), respectively. Despite the general assumption that local anesthetic binding to the solid membrane is negligible, this study showed that the solid membrane binding amounts to 57.5% of the liquid membrane binding. Binding of the charged form to the liquid or solid membrane was not detectable under the present experimental condition of 0.03 mM tetracaine bulk concentration. The present method measures metachromasia of local anesthetics when bound to lipid membranes. Its advantage is that the separation of the vesicles from the solution is not required. A linearized equation is presented that estimates the partition coefficient or binding constant graphically from a linear plot of the absorbance data. The method is applicable for estimation of drug partition when a measurable spectral change occurs due to complex formation.

Buffers↗

PubMed1988

Two Ca2+-dependent ATPases in rat liver plasma membrane. The previously purified (Ca2+-Mg2+)-ATPase is not a Ca2+-pump but an ecto-ATPase.

We have shown that the rat liver plasma membrane has at least two (Ca2+-Mg2+)-ATPases. One of them has the properties of a plasma membrane Ca2+-pump (Lin, S.-H. (1985) J. Biol. Chem. 260, 7850-7856); the other one, which we have purified (Lin, S.-H., and Fain, J.N. (1984) J. Biol. Chem. 259, 3016-3020) and characterized (Lin, S.-H. (1985) J. Biol. Chem. 260, 10976-10980) has no established function. In this study we present evidence that the purified (Ca2+-Mg2+)-ATPase is a plasma membrane ecto-ATPase. In hepatocytes in primary culture, we can detect Ca2+-ATPase and Mg2+-ATPase activities by addition of ATP to the intact cells. The external localization of the active site of the ATPase was confirmed by the observation that the Ca2+-ATPase and Mg2+-ATPase activities were the same for intact cells, saponin-treated cells, and cell homogenates. Less than 14% of total intracellular lactate dehydrogenase, a cytosolic enzyme, was released during a 30-min incubation of the hepatocytes with 2 mM ATP. This indicates that the hepatocytes maintained cytoplasmic membrane integrity during the 30-min incubation with ATP, and the Ca2+-ATPase and Mg2+-ATPase activity measured in the intact cell preparation was due to cell surface ATPase activity. The possibility that the ecto-Ca2+-ATPase and Mg2+-ATPase may be the same protein as the previously purified (Ca2+-Mg2+)-ATPase was tested by comparing the properties of the ecto-ATPase with those of (Ca2+-Mg2+)-ATPase. Both the ecto-ATPase and the (Ca2+-Mg2+)-ATPase have broad nucleotide-hydrolyzing activity, i.e. they both hydrolyze ATP, GTP, UTP, CTP, ADP, and GDP to a similar extent. The effect of Ca2+ and Mg2+ on the ecto-ATPase activity is not additive indicating that both Ca2+- and Mg2+-ATPase activities are part of the same enzyme. The ecto-ATPase activity, like the (Ca2+-Mg2+)-ATPase, is not sensitive to oligomycin, vanadate, N-ethylmaleimide and p-chloromercuribenzoate; and both the ecto-ATPase and purified (Ca2+-Mg2+)-ATPase activities are insensitive to protease treatments. These properties indicate that the previously purified (Ca2+-Mg2+)-ATPase is an ecto-ATPase and may function in regulating the effect of ATP and ADP on hepatocyte Ca2+ mobilization (Charest, R., Blackmore, P.F., and Exton, J.H. (1985) J. Biol. Chem. 260, 15789-15794).

Adenosine Triphosphatases↗

PubMed1988

Effect of surface ionization of dimyristoylphosphatidic acid vesicle membranes on the main phase-transition enthalpy and temperature.

The main phase transition of phospholipid bilayers is a property expressed by the order-disorder conformational change of the lipid tails. Nevertheless, with ionizable phospholipids, changes in the surface charge have large effects on the membrane properties. The free energy of a charged phospholipid membrane depends on the degree of ionization, area per phospholipid molecule, and the temperature. Here, the effect of surface electrostatic charges on the temperature and the enthalpy of the main phase transition of dimyristoylphosphatidic acid vesicle membranes is analyzed. A simple equation is presented that describes the relationship among the surface charge density, the phase-transition temperature, the surface area ratio between solid and liquid membranes, and the excess enthalpy. The theory indicated that the pH-induced shift in the excess enthalpy is attributable to the change in the surface area ratio between the solid and liquid membranes.

Calorimetry, Differential Scanning↗

PubMed1988

Steady-state kinetics of hydrolysis of dansyl-peptide substrates by leucine aminopeptidase.

Stopped-flow fluorescence experiments have been carried out at 23 degrees C to study the hydrolysis of Leu-Gly-NHNH-Dns [Dns = 5-(dimethylamino)naphthalene-1-sulfonyl] and Leu-Gly-NH(CH2)2NH-Dns by porcine kidney cytosol leucine aminopeptidase (LAP). Experiments have been performed with LAP species containing Mg(II), Mn(II), Ni(II), Cu(II), Zn(II), and no metal ion at the regulatory metal binding site. The fluorescence changes observed on hydrolysis of these dansyl substrates by LAP arise from changes in the concentration of substrate. Several kinetic relationships have been developed that allow the steady-state kinetic parameters for these reactions to be determined from the stopped-flow fluorescence traces. When any of the five metal ions are bound at the regulatory site, kcat and KM are both raised to approximately the same extent with the result that the maximum increase observed for kcat/KM is only approximately twofold. The effects of these metal ions on kcat, KM, and kcat/KM observed for these substrates differ markedly from those for less physiologically relevant substrates, such as Leu-p-nitroanilide, that do not have amino acids on both sides of the scissile bond. This suggests that earlier conclusions regarding the effect of the regulatory metal ion on the activity of LAP may have been misleading and casts doubt as to whether the term "regulatory site" has validity in the context of LAP-catalyzed reactions under physiological conditions.

Algorithms↗

PubMed1988

Stopped-flow cryoenzymological investigation of the pre-steady-state kinetics of hydrolysis of Leu-Gly-NHNH-Dns by leucine aminopeptidase.

Stopped-flow fluorescence experiments have been carried out to study the steady-state kinetics of hydrolysis of Leu-Gly-NHNH-Dns [Dns = 5-(dimethylamino)naphthalene-1-sulfonyl] by porcine kidney cytosol leucine aminopeptidase (LAP) in 50% v/v methanol/buffer solution at ambient temperature and the pre-steady-state kinetics of this reaction in the -35 to 0 degrees C temperature range. Experiments have been carried out on LAP species containing Mg(II), Mn(II), Cu(II), Ni(II), Zn(II), and no metal ion at the regulatory metal binding site. At ambient temperatures, the stopped-flow fluorescence changes observed on hydrolysis of the substrate have been used to measure the steady-state kinetic parameters kcat and KM. The results show that 50% v/v methanol lowers the values of kcat from 2- to 12-fold compared to the reactions in the absence of methanol for all of the metallo-LAP, but that the values of KM are essentially unaffected. The pre-steady-state reactions carried out under nonturnover conditions at -35 degrees C reveal a new relaxation for LAP species with Ni(II), Cu(II), and Zn(II) in the regulatory site. The value of kobsd for this relaxation reaches a plateau at high substrate concentrations, and the magnitude of its fluorescence change at a fixed concentration of substrate is proportional to the enzyme concentration. Thus, this relaxation corresponds to the production and decay of a new enzyme-substrate intermediate not observed at higher temperatures whose fluorescence differs from that of the succeeding intermediate that is normally seen above -26 degrees C.

Algorithms↗

PubMed1988

Resonance Raman evidence for an unusually strong exogenous ligand-metal bond in a monomeric nitrosyl manganese hemoglobin.

Resonance Raman spectroscopy has been employed to determine the vibrational modes of monomeric nitrosyl manganese Chironomus thummi thummi hemoglobin (CTT IV). This insect hemoglobin has no distal histidine. By applying various isotope-labeled nitric oxides (14N16O, 15N16O, 14N18O), we have identified the Mn11-NO stretching model at 628 cm-1, the Mn11-N-O bending mode at 574 cm-1 and the N-O stretching mode at 1735 cm-1. The results suggest a strong Mn11-NO bond and a weak N-O bond. The vinyl group substitution does not influence the nu (Mn11-NO), delta (Mn11-N-O) and nu (N-O) vibrations. The Mn11-NO stretching frequency is insensitive to distal histidine interactions with NO, whereas the N-O stretching frequency is sensitive. Nitric oxide also binds to Met manganese CTT IV to form an Mn111. NO complex which undergoes a slow but complete autoreduction resulting in the Mn11.NO species. In manganese meso-IX CTT IV, the Mn111. NO Mn11. NO conversion alters the intensities of the porphyrin ring modes at 342, 360, 1587 and 1598 cm-1, but shifts the frequencies at 1504 and 1633 cm-1 (in Mn111.NO) to 1497 and 1630 cm-1 (in Mn11. NO), respectively. The unshifted marker line at 1378 cm-1 reflects the fact that the pi electron densities of the porphyrin ring are the same in the two complexes.

Animals↗

PubMed1988