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Biomedical subjects

R L Pan

Publications and source records attributed to R L Pan.

35 records · Page 2Linked to original sources
PubMed1992

Inhibition of tonoplast ATPase from etiolated mung bean seedlings by fluorescein 5'-isothiocyanate.

Fluorescein 5'-isothiocyanate (FITC) was used to modify the lysine residue in the active site of tonoplast H(+)-ATPase from etiolated mung-bean (Vigna radiata L.) seedlings. FITC caused marked inactivation of the enzyme activities of both membrane-bound and soluble ATPase and its associated H+ translocation. The SDS/PAGE pattern revealed that the FITC-binding site was in the large (A) subunit of ATPase. Inhibition could be substantially prevented by its physiological substrate ATP, pyrophosphate and nucleotides in the decreasing order: ATP greater than pyrophosphate greater than ADP greater than AMP greater than GTP greater than CTP greater than UTP. The mode of inhibition by FITC was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first-order kinetics with a Ki of 0.33 mM, a minimum inactivation half-time of 110 s, and a first-order rate constant of 0.244 s-1. A double-logarithmic plot of apparent rate constant versus FITC concentration gave a slope of 0.913, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labelling studies indicated that the incorporation of approx. 1 mol of FITC/mol of ATPase is sufficient to inhibit ATPase completely. The enhancement and blue shift of emission maxima of FITC after modification of ATPase indicated that the labelled lysine residue was located in a relatively hydrophobic domain.

Adenosine Triphosphatases↗
PubMed1992

Inhibition of tonoplast ATPase by 2',3'-dialdehyde derivative of ATP.

The 2',3'-dialdehyde derivative of ATP (dial-ATP) has been shown to be an affinity label for the ATP binding site of the H(+)-ATPase from tonoplast of etiolated mung bean seedlings (Vigna radiata L.). The dial-ATP caused marked inactivation of enzymatic activities of both membrane-bound and soluble ATPase and its associated proton translocation. The inactivation was reversible, but could be stabilized by NaBH(4). The sodium dodecyl sulfatepolyacrylamide gel electrophoresis pattern revealed that the dial-ATP binding site was in the large (A) subunit of ATPase. The inhibition could be substantially protected by its physiological substrate ATP, pyrophosphate, and nucleotides in the decreasing order: ATP > pyrophosphate > ADP = AMP > GTP > CTP = UTP. A Lineweaver-Burk plot showed that the mode of inhibition was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first order kinetics with a K(i) of 4.1 millimolar, a minimum inactivation half-time of 20 seconds, and a pseudo-first order rate constant of 0.035 s(-1). The double logarithmic plot of apparent rate constant versus dial-ATP concentration gave a slope of 0.927, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labeling studies with [(3)H]dial-ATP indicate that the incorporation of approximately 1 mole of dial-ATP per mole ATPase is sufficient to completely inhibit the ATPase. A working model of nonequivalent subunits for enzymatic mechanism of vacuolar ATPase is suggested.

Journal Article↗
PubMed1991

Functional size analysis of pyrophosphatase from Rhodospirillum rubrum determined by radiation inactivation.

A radiation inactivation technique was employed to determine the functional size of pyrophosphatase (PPase) from the chromatophores of Rhodospirillum rubrum. The activities of hydrolysis and synthesis reactions of pyrophosphatase and its coupled proton translocation decayed in a simple exponential function with the increase of radiation dosages. D37 values of 5.2 +/- 0.7 and 5.8 +/- 0.8 Mrads were obtained for pyrophosphate hydrolysis and its associated proton translocation yielding molecular masses of 167.7 +/- 30.7 and 156.3 +/- 26.6 kDa, respectively. Similarly, a D37 value of 4.4 +/- 0.6 Mrads was measured for the acid-base induced pyrophosphate synthesis resulting in a radiation sensitive size of 196.3 +/- 31.9 kDa.

Hydrolysis↗
PubMed1991

Multivariate comparison of the forelimb between stump-tailed and rhesus macaques.

Rhesus macaques and stump-tailed macaques are sympatric in western Yunnan (China), coexisting or occupying habitats that show little difference. This paper tests hypotheses based on theoretical expectation from the differing biomechanical demands of terrestrial and arboreal quadrupedalism in stump-tailed macaques and rhesus macaques, respectively. Individuals of these two macaque taxa were markedly separated by the first two principal components and discriminant analyses based on 18 variables of the upper limb. The rhesus macaques appear to be more adapted for arboreal quadruped habits because of elongation of the clavicle and forearm, a larger humeral head and greater midshaft sagittal diameters of the radius and ulna.

Analysis of Variance↗
PubMed1990

An essential arginyl residue in the tonoplast pyrophosphatase from etiolated mung bean seedlings.

Tonoplast membrane of etiolated mung bean (Vinga radiata. L.) seedlings contained H(+)-translocating pyrophosphatase (PPase). Modification of tonoplast vesicles and partially purified PPase from etiolated mung bean seedlings with arginine-specific reagents, phenylglyoxal (PGO) and 2,3-butanedione (BD), resulted in a marked decline in H(+)-translocating PPase activity. The half-maximal inhibition was brought about by 20 millimolar PGO and 50 millimolar BD for membrane bound and 1.5 millimolar PGO and 5.0 millimolar BD for soluble PPase, respectively. The substrate, Mg(2+)-pyrophosphate, provided partial protection against inactivation by these reagents. Loss of activity of partially purified PPase followed pseudo-first order kinetics. The double logarithm plots of pseudo-first order rate constant versus reagent concentrations gave slopes of 0.88 (PGO) and 0.90 (BD), respectively, suggesting that the inactivation may possibly result from reaction of at least one arginyl residue at the active site of H(+)-translocating PPase.

Journal Article↗
PubMed1989

Purification and characterization of tonoplast ATPase from etiolated mung bean seedlings.

The tonoplast ATPase from etiolated seedlings of Vigna radiata L. (mung bean) was isolated using a two-step detergent solubilization modified from Mandala and Taiz (S Mandala, L Taiz [1985] Plant Physiol 78: 327-333). After ultracentrifugation on 10 to 28% sucrose gradient, the ATPase showed a 31.6-fold purification over the initial specific activity of the starting tonoplast-enriched membranes. The purified ATPase used Mg(2+)-ATP as the preferred substrate. The tonoplast ATPase was isolated in a form with characteristics similar to that on its native membrane environment. Analysis by SDS-PAGE revealed two prominent bands with molecular weights of 78,000 (alpha subunit) and 64,000 (beta subunit). The intensity of Coomassie blue staining showed a 1:1 stoichiometry for alpha and beta subunits. The amino acid composition of alpha and beta subunits also confirmed the suggested stoichiometry of the subunit composition of the tonoplast ATPase. Moreover, radiation inactivation analysis yielded a functional size of 414 +/- 24 and 405 +/- 25 kilodaltons for soluble and membrane bound tonoplast ATPases, respectively. It is possible that the functioning tonoplast ATPase may be in a form of alphabeta-heteromultimer.

Journal Article↗
PubMed1988

Radiation inactivation analysis of chloroplast CF0-CF1 ATPase.

Radiation inactivation technique was employed to measure the functional size of adenosine triphosphatase of spinach chloroplasts. The functional size for acid-base-induced ATP synthesis was 450 +/- 24 kilodaltons; for phenazine methosulfate-mediated ATP synthesis, 613 +/- 33 kilodaltons; and for methanol-activated ATP hydrolysis, 280 +/- 14 kilodaltons. The difference (170 +/- 57 kilodaltons) between 450 +/- 24 and 280 +/- 14 kilodaltons is explained to be the molecular mass of proton channel (coupling factor 0) across the thylakoid membrane. Our data suggest that the stoichiometry of subunits I, II, and III of coupling factor 0 is 1:2:15. Ca2+- and Mg2+-ATPase activated by methanol, heat, and trypsin digestion have a similar functional size. However, anions such as SO3(2-) and CO3(2-) increased the molecular mass for both ATPase's (except trypsin-activated Mg2+-ATPase) by 12-30%. Soluble coupling factor 1 has a larger target size than that of membrane-bound. This is interpreted as the cold effect during irradiation.

Adenosine Triphosphatases↗
PubMed1987

Functional size of photosynthetic electron transport chain determined by radiation inactivation.

Radiation inactivation technique was employed to determine the functional size of photosynthetic electron transport chain of spinach chloroplasts. The functional size for photosystem I+II (H(2)O to methylviologen) was 623 +/- 37 kilodaltons; for photosystem II (H(2)O to dimethylquinone/ferricyanide), 174 +/- 11 kilodaltons; and for photosystem I (reduced diaminodurene to methylviologen), 190 +/- 11 kilodaltons. The difference between 364 +/- 22 (the sum of 174 +/- 11 and 190 +/- 11) kilodaltons and 623 +/- 37 kilodaltons is partially explained to be due to the presence of two molecules of cytochrome b(6)/f complex of 280 kilodaltons. The molecular mass for other partial reactions of photosynthetic electron flow, also measured by radiation inactivation, is reported. The molecular mass obtained by this technique is compared with that determined by other conventional biochemical methods. A working hypothesis for the composition, stoichiometry, and organization of polypeptides for photosynthetic electron transport chain is proposed.

Journal Article↗
PubMed1986

The two binding sites for DCMU in Photosystem II.

Measurements on the fluorescence induction of Triton X-100 extracted Photosystem II (PSII) particles confirmed the existence of the two sites of inhibition in PSII for the herbicide DCMU. The two sites were located on the reducing and oxidizing sides of PSII, respectively. The inhibition on the oxidizing side, unlike that on the reducing side which was of the "none or all" type, was found only to slow down the electron donation at low concentrations of DCMU. The results also suggested that the inhibitions of DCMU at these two sites were mutually exclusive, i.e., the binding on one site prevented the binding on the other site.

Binding Sites↗
PubMed1985

The high affinity binding site for calcium on the oxidizing side of photosystem II.

Preparations of photosystem II complex from spinach chloroplasts with Triton X-100 were treated with 1 M KCl to release 17 KDa and 23 KDa polypeptides. The inhibited oxygen evolution activity could be reactivated by adding high concentration (mM) of Ca++ or by reconstituting 17 KDa and 23 KDa polypeptides which were found to promote high affinity binding of Ca++ to the reconstituted membranes (Ghanotakis et al. FEBS (1984) 170, 169-173). Inclusion of 50 mM Ca++ during KCl treatment did not prevent the release of 17 KDa and 23 KDa polypeptides but protected oxygen evolution from being inactivated. It is explained by preservation of the high affinity binding site for Ca++ if, Ca++ is present during the salt treatment even though depletion of 17 KDa and 23 KDa polypeptides usually results in replacement by a low affinity (mM) binding site for Ca++. It also implies that the high affinity binding site is not located on 17 KDa and 23 KDa polypeptides.

Binding Sites↗
PubMed1985

Conformational changes in plastocyanin.

The visible and near-uv absorption and circular dichroic spectra were determined for spinach and poplar plastocyanin under a variety of conditions. The visible spectra showed that the copper center was invariant to changes in species, chemical modification with ethylenediamine, and addition of high concentrations of salt [2.7 M (NH4)2SO4]. In contrast, the near-uv spectra were sensitive to these conditions. Reduction of plastocyanin also altered its near-uv absorption and circular dichroic spectra. It is unlikely that these spectral changes were due to charge transfer bands since the near-uv CD spectrum of apo-plastocyanin was almost identical to that of reduced plastocyanin. There were no corresponding changes in the far-uv spectra which monitor protein secondary structure. The most likely explanation is that the protein has a flexible tertiary conformation. Conformational changes may be important in regulating electron transport. If plastocyanin is a mobile electron carrier, differential binding of the oxidized and reduced forms of plastocyanin to its reaction partners cytochrome f and P700 could facilitate electron transport.

Apoproteins↗
PubMed1979

Photosystem II energy coupling in chloroplasts with H2O2 as electron donor.

NH2OH-treated, non-water-splitting chloroplasts can oxidize H2O2 to O2 through Photosystem II at substantial rates (100--250 muequiv . h-1 . mg-1 chlorophyll with 5 mM H2O2) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H2O2 leads to Photosystem II leads to dimethylquinone reaction supports phosphorylation with a P/e2 ratio of 0.25--0.35 and proton uptake with H+/e values of 0.67 (pH 8)--0.85 (pH 6). These are close to the P/e2 value of 0.3--0.38 and the H+/e values of 0.7--0.93 found in parallel experiments for the H2O leads to Photosystem II leads to dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor leads to Photosystem II leads to dibromothymoquinone (leads to O2) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I-, ferrocyanide).

Chloroplasts↗
PubMedDate not supplied

Maintenance of human sperm motility and prevention of oxidative damage through co-culture incubation.

Co-culture incubation is one of the important techniques used in basic and clinical research of assisted reproduction. In this study, sperm samples from 40 healthy donors were prepared for co-culture incubation with Vero cells which had been derived from the kidney fibroblasts of the African green monkey, Cercopithecus aethiops. We found that the motility characteristics of ejaculated human sperm co-cultured with Vero cells were largely maintained and the percentage of hyperactivated sperm in the co-culture group was not affected. While the sperm of the control group completely lost the motility at 12 h incubation at 37 degrees C in 5% CO2, the sperm co-cultured with Vero cells still maintained 74% of the original motility. Lipid peroxidation and accumulation of 8-hydroxy-2'-deoxyguanosine in spermatozoa were also reduced by the co-culture incubation, which strongly indicates that intercellular interactions may play some role in the maintenance of sperm functions. We conclude that the oxidative damage in vitro of the sperm can be reduced by the co-culture system and thereby maintains the function of sperm from oxidative damage.

8-Hydroxy-2'-Deoxyguanosine↗
PubMedDate not supplied

Direct measurement of the tail beat frequency of human sperm by flash light synchronization.

Based on the synchronization between a periodic vibration and flashing light, we designed a device to determine the flagellar beating frequency of human sperm. The head of a spermatozoon was either held by the tip of a micropipette operated with a micromanipulator or adhered by itself on the surface of a glass slide when the sperm swam out of the micropipette into the fresh Ham's F-10 medium. The beat frequency of the flagellum was measured by synchronization of the frequency-adjustable flash light built on an inverted microscope. The light frequency synchronizer included a controller, a pulse generator, a signal counter, and a flash illuminator. During each measurement, the spermatozoon was transferred to the center of the observing field and the frequency generator created a series of signals which developed flashing signals onto the sample plane. When the vibration of the flagellum was observed as a constant two-step-like movement after frequency adjustment, the beating frequency was read from the signal counter and the count was twice that of the beat frequency of the sperm tail. As the flash signal was decreased to half of this frequency, an apparently immobilized sperm flagellum was observed and the exact beating frequency could then be determined. This device was then used to measure the effect of pentoxifylline on sperm motility. The results showed that the increase of tail beat frequency as measured by this device is well correlated with the increase of beat cross frequency as detected by a computer-assisted semen analyzer. These findings suggest that this flash light synchronization device is a reliable and useful system for the assessment of sperm motility.

Humans↗