Dipeptide uptake by adenohypophysial folliculostellate cells

1996 ◽  
Vol 271 (1) ◽  
pp. C210-C217 ◽  
Author(s):  
C. Otto ◽  
S. tom Dieck ◽  
K. Bauer
Keyword(s):  
Primary Cultures ◽  
Maximum Velocity ◽  
Peptide Transporter ◽  
Michaelis Constant ◽  
Folliculostellate Cells ◽  
S 100 ◽  
Microscopic Studies ◽  
Energy Dependent ◽  
Dipeptide Uptake ◽  
Dipeptide Derivative

Dipeptide uptake was studied in primary cultures from rat anterior pituitaries by use of radiolabeled carnosine and the fluorescent dipeptide derivative beta-Ala-Lys-N epsilon-AMCA (AMCA is 7-amino-4-methylcoumarin-3-acetic acid). Fluorescence microscopic studies revealed that the reporter peptide specifically accumulated in the S-100 positive folliculostellate cells that do not produce any known hormone. The dipeptide derivative was taken up in unmetabolized form by an energy-dependent saturable process with apparent kinetic constants as follows: Michaelis constant, 19 microM; maximum velocity, 5.5 nmol.mg protein-1.h-1. This high-affinity transporter was strongly affected by inhibitors of sodium/proton exchangers and thus appeared to be driven by a proton gradient. Competition studies revealed that the peptide transporter exhibits broad substrate specificity with a preference for hydrophobic dipeptides. In contrast to free amino acids and the pseudotetrapeptide amastatin, tripeptides were also accepted. Compounds without an alpha- and beta-amino group, such as captopril, thiorphan, and benzylpenicillin, did not affect uptake of the reporter peptide, although they were substrates of the well-characterized intestinal and renal dipeptide transporters.

Functional linkage of H+/peptide transporter PEPT2 and Na+/H+ exchanger in primary cultures of astrocytes from mouse cerebral cortex

2005 ◽  
Vol 1044 (1) ◽  
pp. 33-41 ◽  
Author(s):  
Miyuki Wada ◽  
Sakiko Miyakawa ◽  
Ayumi Shimada ◽  
Naoki Okada ◽  
Akira Yamamoto ◽  
...  
Keyword(s):  
Cerebral Cortex ◽  
Primary Cultures ◽  
Peptide Transporter ◽  
Functional Linkage ◽  
Mouse Cerebral Cortex

Mechanisms of potassium transfer across the dually perfused rat placenta

1993 ◽  
Vol 265 (2) ◽  
pp. R341-R347 ◽  
Author(s):  
T. Mohammed ◽  
J. Stulc ◽  
J. D. Glazier ◽  
R. D. Boyd ◽  
C. P. Sibley
Keyword(s):  
Diffusion Coefficient ◽  
Maximum Velocity ◽  
T Test ◽  
Michaelis Constant ◽  
Diffusion Limited ◽  
51Cr Edta ◽  
The Asymmetry ◽  
Paired T Test

The purpose of this study was to directly investigate the mechanisms of K+ transfer across the rat placenta, which was isolated and perfused through both its maternal and fetal circulations. Unidirectional maternofetal (Kmf) and fetomaternal (Kfm) clearances for 42K, 51Cr-labeled EDTA (used as a diffusion-limited paracellular marker), and 3H2O (used as a flow-limited marker) were respectively 232 +/- 36, 12 +/- 4, and 1,020 +/- 260 (mf) and 96 +/- 26, 18 +/- 6, and 737 +/- 176 (fm) microliters.min-1 x g placenta-1. Calculated K+ fluxes were asymmetric, being 0.75 +/- 0.12 and 0.41 +/- 0.12 mumol.min-1 x g placenta-1 for maternofetal and fetomaternal, respectively (mean +/- SE, n = 6; P < 0.01, paired t test). Although Kmf for 3H2O was 28% higher than Kfm, this could not completely account for the asymmetry in K+ fluxes. Kmf for 42K was 12-70 times higher than that for 51Cr-EDTA (presumed to be a paracellular marker), although its diffusion coefficient is only 2.5 times higher. An apparent Michaelis constant (Km) of 11.0 +/- 2.4 mM and maximum velocity (Vmax) of 3.8 +/- 0.33 mumol.min-1 x g placenta-1 was calculated by Michaelis-Menten analysis of the transcellular component of maternofetal flux (Jmf) for K+. Ouabain or barium (1 mM in maternal and fetal perfusate) reduced Kmf for 42K from 250 +/- 38 to 76 +/- 13 microliters.min-1 x g placenta-1 (n = 4; P < 0.01) and from 358 +/- 31 to 106 +/- 18 microliters.min-1 x g placenta-1 (n = 5; P < 0.001). Neither drug had any effect on Kmf for 51Cr-EDTA or 3H2O.(ABSTRACT TRUNCATED AT 250 WORDS)

Copper transport and kinetics in cultured C6 rat glioma cells

1995 ◽  
Vol 269 (4) ◽  
pp. C892-C898 ◽  
Author(s):  
Y. Qian ◽  
E. Tiffany-Castiglioni ◽  
E. D. Harris
Keyword(s):  
Membrane Fraction ◽  
Glioma Cells ◽  
Maximum Velocity ◽  
C6 Cells ◽  
Michaelis Constant ◽  
Rat Glioma ◽  
Saturation Kinetics ◽  
Efflux Mechanism ◽  
Import And Export ◽  
P Type

C6 rat glioma cells accumulate and efflux 67Cu. Both processes showed saturation kinetics with increasing 67Cu concentration. The Michaelis constant (Km) for uptake was 0.63 +/- 0.14 microM; maximum velocity (Vmax) was 3.29 +/- 0.57 pmol Cu.mg protein-1.min-1. The Km for efflux was 0.15 +/- 0.06 microM; Vmax was 1.08 +/- 0.71 pmol Cu.mg protein-1.min-1. p-Chloromercuribenzoate (p-CMB) totally blocked 67Cu efflux but had no effect on Km or Vmax of uptake. Total 67Cu in the cell after 50 min was partitioned equally between particulate and soluble fractions. p-CMB-treated cells accumulated more 67Cu, but < 10% was bound to the particulate (membrane) fraction. Pb also increased 67Cu accumulation without affecting Km and Vmax of 67Cu uptake. These data suggest that carriers for import and export of Cu in C6 cells are distinct or operate in two different cellular environments. Efflux is a sulfhydryl-dependent process subject to inhibition by Pb. The data are consistent with a P-type ATPase in the efflux of Cu from cells and the potential for Pb to inhibit the efflux mechanism.

Heart, liver, and skeletal muscle myeloperoxidase activity during exercise

1996 ◽  
Vol 80 (4) ◽  
pp. 1331-1335 ◽  
Author(s):  
A. N. Belcastro ◽  
G. D. Arthur ◽  
T. A. Albisser ◽  
D. A. Raj
Keyword(s):  
Skeletal Muscle ◽  
Contractile Activity ◽  
Maximum Velocity ◽  
Neutrophil Infiltration ◽  
Exercise Group ◽  
Rat Tissues ◽  
Myeloperoxidase Activity ◽  
Michaelis Constant ◽  
Similar Range ◽  
Gastrocnemius Muscles

The purpose of this study was to determine whether contractile activity associated with running exercise was a prerequisite for neutrophil infiltration into rat tissues. H2O2-dependent myeloperoxidase (MPO) activity for rat (n = 8) liver, heart, and gastrocnemius muscles was assayed after 58 +/- 11 min of running to voluntary exhaustion (25 m/min; 0% grade). MPO activity values measured with 0.6 mM H2O2 were 0.988 +/- 0.331 (SD) U/g (skeletal muscle), 1.563 +/- 0.303 U/g (heart), and 1.652 +/- 0.510 U/g (liver) for control samples, compared with 1.690 +/- 0.321, 3.128 +/- 1.221, and 2.752 +/- 0.437 U/g, respectively, for the exercise group (P < or = 0.05). Kinetic analysis revealed that maximum velocity for all tissues increased as a result of the exercise (P < 0.05). The Michaelis constant (Km) values at rest for all tissues were similar (range 0.53-0.57 mM H2O2; P = or = 0.05). Exercise did not alter the Km values for cardiac and liver samples; however, for skeletal muscle, the Km was 28% lower than control (P < or = 0.05). The results of this study show that, with prolonged running, MPO activity is elevated in most rat tissues and not exclusively in skeletal muscle. Moreover, the metabolic status of the tissues may be an important factor for neutrophil infiltration with exercise and not exclusively the type of muscle contraction, as previously hypothesized.

scholarly journals The temperature-dependence of adenylate cyclase from baker's yeast

1979 ◽  
Vol 181 (3) ◽  
pp. 539-543 ◽  
Author(s):  
J Londesborough ◽  
K Varimo
Keyword(s):  
Reaction Mechanism ◽  
Adenylate Cyclase ◽  
Temperature Dependence ◽  
Reaction Rate ◽  
Arrhenius Plot ◽  
Maximum Velocity ◽  
Michaelis Constant ◽  
Arrhenius Plots ◽  
Temperature Range ◽  
Membrane Bound

The Michaelis constant of membrane-bound adenylate cyclase increased from 1.1 to 1.8 mM between 7 and 38 degrees C (delta H = 13 kJ/mol). Over this temperature range, the maximum velocity increased 10-fold, and the Arrhenius plot was nearly linear, with an average delta H* of 51 kJ/mol. The temperature-dependence of the reaction rate at 2 mM-ATP was examined in more detail: for Lubrol-dispersed enzyme, Arrhenius plots were nearly linear with average delta H* values of 45 and 68 kJ/mol, respectively, for untreated and gel-filtered enzymes; for membrane-bound enzyme, delta H changed from 40 kJ/mol above about 21 degrees C to 62 kJ/mol below 21 degrees C, but this behaviour does not necessarily indicate an abrupt, lipid-induced, transition in the reaction mechanism.

Na(+)-dependent sulfate transport in opossum kidney cells is DIDS sensitive

1993 ◽  
Vol 265 (1) ◽  
pp. C54-C61 ◽  
Author(s):  
H. S. Tenenhouse ◽  
J. Martel
Keyword(s):  
Maximum Velocity ◽  
Hill Coefficient ◽  
Disulfonic Acid ◽  
Kidney Cells ◽  
Sulfate Transport ◽  
Sulfate Uptake ◽  
Michaelis Constant ◽  
Opossum Kidney ◽  
Renal Brush Border Membrane ◽  
Opossum Kidney Cells

Sulfate transport was examined in OK/E cells, a clonal subline of opossum kidney cells that express several differentiated functions of the proximal tubule. Extracellular Na+ stimulated [35S]sulfate uptake five- to sixfold. Hill analysis demonstrating the dependence of sulfate uptake on Na+ concentration yielded a Hill coefficient of 1.5 and a Michaelis constant (KNa+) of 23 mM. Na(+)-dependent sulfate uptake was increased by lowering the pH from 7.4 to 6.4, decreased by raising the pH to 8.4 and inhibited by a 10-fold molar excess of SO3(2-), S2O3(2-) and CrO4(2-), but not by phosphate. The Na(+)-mediated component of sulfate uptake was saturable and kinetic parameters were estimated [Michaelis constant (Km) = 2.4 +/- 0.2 mM and maximum velocity (Vmax) = 125 +/- 15 pmol.mg protein-1.min-1]. Omitting extracellular Cl- resulted in a significant increase in the affinity of the carrier for sulfate (Km = 0.5 mm), without changing Vmax, consistent with competitive inhibition by Cl-. Na(+)-dependent sulfate uptake in OK/E cells was also inhibited by HCO3- [half-maximal inhibitory concentration (IC50) = 7 mM], 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, IC50 = 0.9 microM), 0.5 mM picrylsulfonic acid and 0.1 mM ethacrynic acid, but not by 1 mM amiloride. Na(+)-dependent, DIDS-sensitive sulfate uptake was also expressed in the parental OK cell line and was not influenced by serum or 3,3',5-triiodo-L-thyronine. We conclude that Na(+)-dependent sulfate uptake in OK/E cells observes many of the features of Na(+)-sulfate cotransport in the renal brush-border membrane and provides a useful model to investigate the regulation of renal sulfate transport.

Control of H(+)-HCO3- plasma membrane transporters by urea hyperosmolality in rat medullary thick ascending limb

1994 ◽  
Vol 266 (5) ◽  
pp. C1157-C1164 ◽  
Author(s):  
F. Leviel ◽  
M. Froissart ◽  
H. Soualmia ◽  
J. Poggioli ◽  
M. Paillard ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Calcium Concentration ◽  
Inhibitory Concentration ◽  
Initial Rate ◽  
Maximum Velocity ◽  
Membrane Transporters ◽  
Thick Ascending Limb ◽  
Michaelis Constant ◽  
Medullary Thick Ascending Limb ◽  
Experimental Protocols

Hyperosmolality inhibits bicarbonate absorption by the rat medullary thick ascending limb (MTAL) by unknown mechanisms. Intracellular pH (pHi) was monitored with use of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein in rat MTAL tubule suspensions to specify the H(+)-HCO3- membrane transporters affected by hyperosmolality. Measurements were made after > or = 15-min incubation of the cells in media rendered hypertonic by urea to avoid any change in cell volume. Na(+)-H+ antiport activity, estimated from the Na(+)-induced initial rate of pHi recovery of Na(+)-depleted acidified cells in the presence of 0.1 mM furosemide to inhibit Na(+)-K(+)-2Cl- cotransport, was inhibited by 300 mM urea and 10(-8) M arginine vasopressin (AVP) in an additive manner. Na(+)-H+ antiport inhibition by urea hyperosmolality was maximal at 300 mM urea with a half-maximal inhibitory concentration of 75 mM and was due to a 28% decrease in maximum velocity (Vmax) with no effect on the Michaelis constant for sodium. Urea hyperosmolality (300 mM) did not affect steady-state intracellular calcium concentration ([Ca2+]i), assessed with use of fura 2 fluorescence, and still inhibited Na(+)-H+ antiport in MTAL cells loaded with 1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid to minimize any transient change in [Ca2+]i during the preincubation in urea medium. Furthermore, 300 mM urea did not stimulate basal or AVP-induced adenosine 3',5'-cyclic monophosphate (cAMP) accumulation. Plasma membrane H(+)-adenosinetriphosphatase (ATPase) activity and HCO3- transport, assessed by appropriate experimental protocols, were unaltered by 300 mM urea.(ABSTRACT TRUNCATED AT 250 WORDS)

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