scholarly journals Variable H+/substrate stoicheiometries in Rhodotorula gracilis are caused by a pH-dependent protonation of the carrier(s)

1982 ◽  
Vol 208 (2) ◽  
pp. 459-464 ◽  
Author(s):  
R Hauer ◽  
M Höfer
Keyword(s):  
Positive Charge ◽  
Low Ph ◽  
The Other ◽  
Transport Systems ◽  
Lower Affinity ◽  
High Affinity ◽  
High Sugar ◽  
Saturation Kinetics ◽  
Rhodotorula Gracilis ◽  
Ph Dependent

Two carrier-mediated systems transport sugars in the yeast Rhodotorula gracilis depending on the pH. One system, with higher affinity for sugars, catalyses a symport of protons with sugar, whereas the other system, having lower affinity, is independent of protons. This was shown in three different ways. (1) At low pH, where only the high-affinity system works, a H+/sugar stoicheiometry of 1 was found. An increase of the pH and of the sugar concentration, which allowed the low-affinity system to operate, brought about a drop of the stoicheiometry to values below 1. (2) During H+ symport the influx of positive charge was electrically compensated by an equivalent efflux of K+ from the cells. At high pH and high sugar concentrations this stoicheiometry of K+ and sugar decreased concomitant with the H+/sugar stoicheiometry. (3) At pH 7.5 both transport systems were operating, as shown by biphasic saturation kinetics. Under these conditions only the high-affinity transport was found to be electrogenic. These results agree with the theory of an electrogenic H+/sugar symport where changes in the affinity for substrate are brought about by reversible protonation and deprotonation of the carrier.

scholarly journals The STK2 gene, which encodes a putative Ser/Thr protein kinase, is required for high-affinity spermidine transport in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (6) ◽  
pp. 2994-3004 ◽  
Author(s):  
M Kaouass ◽  
M Audette ◽  
D Ramotar ◽  
S Verma ◽  
D De Montigny ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Fusion Gene ◽  
Transport Systems ◽  
Coding Region ◽  
Kinase Gene ◽  
Lower Affinity ◽  
High Affinity ◽  
Polyamine Transport ◽  
Exogenous Spermidine

Eukaryotic polyamine transport systems have not yet been characterized at the molecular level. We have used transposon mutagenesis to identify genes controlling polyamine transport in Saccharomyces cerevisiae. A haploid yeast strain was transformed with a genomic minitransposon- and lacZ-tagged library, and positive clones were selected for growth resistance to methylglyoxal bis(guanylhydrazone) (MGBG), a toxic polyamine analog. A 747-bp DNA fragment adjacent to the lacZ fusion gene rescued from one MGBG-resistant clone mapped to chromosome X within the coding region of a putative Ser/Thr protein kinase gene of previously unknown function (YJR059w, or STK2). A 304-amino-acid stretch comprising 11 of the 12 catalytic subdomains of Stk2p is approximately 83% homologous to the putative Pot1p/Kkt8p (Stk1p) protein kinase, a recently described activator of low-affinity spermine uptake in yeast. Saturable spermidine transport in stk2::lacZ mutants had an approximately fivefold-lower affinity and twofold-lower Vmax than in the parental strain. Transformation of stk2::lacZ cells with the STK2 gene cloned into a single-copy expression vector restored spermidine transport to wild-type levels. Single mutants lacking the catalytic kinase subdomains of STK1 exhibited normal parameters for the initial rate of spermidine transport but showed a time-dependent decrease in total polyamine accumulation and a low-level resistance to toxic polyamine analogs. Spermidine transport was repressed by prior incubation with exogenous spermidine. Exogenous polyamine deprivation also derepressed residual spermidine transport in stk2::lacZ mutants, but simultaneous disruption of STK1 and STK2 virtually abolished high-affinity spermidine transport under both repressed and derepressed conditions. On the other hand, putrescine uptake was also deficient in stk2::lacZ mutants but was not repressed by exogenous spermidine. Interestingly, stk2::lacZ mutants showed increased growth resistance to Li+ and Na+, suggesting a regulatory relationship between polyamine and monovalent inorganic cation transport. These results indicate that the putative STK2 Ser/Thr kinase gene is an essential determinant of high-affinity polyamine transport in yeast whereas its close homolog STK1 mostly affects a lower-affinity, low-capacity polyamine transport activity.

scholarly journals DapE Can Function as an Aspartyl Peptidase in the Presence of Mn2+

2003 ◽  
Vol 185 (16) ◽  
pp. 4748-4754 ◽  
Author(s):  
Daniel H. Broder ◽  
Charles G. Miller
Keyword(s):  
Divalent Cations ◽  
The Other ◽  
Expression Vectors ◽  
Peptidase Activity ◽  
Native Protein ◽  
Lower Affinity ◽  
High Affinity ◽  
High Affinity Site ◽  
Serovar Typhimurium ◽  
Dipeptidase Activity

ABSTRACT Extracts of a multiply peptidase-deficient (pepNABDPQTE iadA iaaA) Salmonella enterica serovar Typhimurium strain contain an aspartyl dipeptidase activity that is dependent on Mn2+. Purification of this activity followed by N-terminal sequencing of the protein suggested that the Mn2+-dependent peptidase is DapE (N-succinyl-l,l-diaminopimelate desuccinylase). A dapE chromosomal disruption was constructed and transduced into a multiply peptidase-deficient (MPD) strain. Crude extracts of this strain showed no aspartyl peptidase activity, and the strain failed to utilize Asp-Leu as a leucine source. The dapE gene was cloned into expression vectors in order to overproduce either the native protein (DapE) or a hexahistidine fusion protein (DapE-His6). Extracts of a strain carrying the plasmid overexpresssing native DapE in the MPD dapE background showed a 3,200-fold elevation of Mn2+-dependent aspartyl peptidase activity relative to the MPD dapE+ strain. In addition, purified DapE-His6 exhibited Mn2+-dependent peptidase activity toward aspartyl dipeptides. Growth of the MPD strain carrying a single genomic copy of dapE on Asp-Leu as a Leu source was slow but detectable. Overproduction of DapE in the MPD dapE strain allowed growth on Asp-Leu at a much faster rate. DapE was found to be specific for N-terminal aspartyl dipeptides: no N-terminal Glu, Met, or Leu peptides were hydrolyzed, nor were any peptides containing more than two amino acids. DapE is known to bind two divalent cations: one with high affinity and the other with lower affinity. Our data indicate that the form of DapE active as a peptidase contains Zn2+ in the high-affinity site and Mn2+ in the low-affinity site.

scholarly journals Characterization of putrescine- and spermidine-transport systems of a rat pancreatic acinar tumoral cell line (AR4-2J)

1990 ◽  
Vol 269 (3) ◽  
pp. 629-632 ◽  
Author(s):  
T G Nicolet ◽  
J L Scemama ◽  
L Pradayrol ◽  
C Seva ◽  
N Vaysse
Keyword(s):  
Cell Line ◽  
Choline Chloride ◽  
The Other ◽  
Transport Systems ◽  
Lower Affinity ◽  
Polyamine Transport ◽  
Tumoral Cell ◽  
Cell Growth And Differentiation ◽  
The Difference

Polyamines are polycationic molecules essential for cell growth and differentiation. Recent work has focused on cell polyamine-transport systems as a way to regulate intracellular polyamine levels. In this study, we demonstrate the presence of two different active transporters for putrescine and spermidine in a rat tumoral cell line (AR4-2J). The first has a Km of 3.1 microM and a Vmax of 3.7 pmol/15 min per micrograms of DNA for putrescine and the second a Km of 0.42 microM and a Vmax of 4.7 pmol/15 min per micrograms of DNA for spermidine. Competition studies performed between the polyamines confirm the difference between these two carriers; one has an equal affinity for the three main polyamines, and the other has a lower affinity for putrescine. Amino acids do not share this transport system, which is Na(+)-independent. Choline chloride inhibits selectively and in a dose-responsive manner the uptake of putrescine without affecting that of spermidine. These data demonstrate that AR4-2J cells possess two polyamine transporters; one is specific for aminopropyl groups (spermidine and spermine), and the other is choline-sensitive, but cannot discriminate between aminobutyl (putrescine) and aminopropyl groups.

Multiple carriers for dipeptide transport: carrier-mediated transport of glycyl-L-proline in renal BBMV

1991 ◽  
Vol 261 (4) ◽  
pp. F670-F678
Author(s):  
H. A. Skopicki ◽  
K. Fisher ◽  
D. Zikos ◽  
R. Bloch ◽  
G. Flouret ◽  
...  
Keyword(s):  
Brush Border Membrane ◽  
Competitive Inhibition ◽  
Membrane Vesicles ◽  
The Other ◽  
Lower Affinity ◽  
High Affinity ◽  
Carrier Mediated Transport ◽  
Transport Carrier ◽  
Dipeptide Transport ◽  
Lower Capacity

To determine whether multiple carriers are responsible for luminal uptake of glycyl-L-proline (Gly-Pro) in the renal proximal tubule, transport of Gly-[3H]Pro was measured in brush-border membrane vesicles (BBMV). A Line-weaver-Burk analysis of Michaelis-Menten kinetics revealed the presence of two carriers: a lower affinity, higher capacity carrier (Km = 1.3 x 10(-2) M; Vmax = 4.6 x 10(-8) mol.mg-1.min-1) and a higher affinity, lower capacity carrier (Km = 2.7 x 10(-7) M; Vmax = 7.8 x 10(-13) mol.mg-1.min-1). The dipeptides Gly-Sar, beta Ala-His, and pyroGlu-His competitively inhibited the low-affinity carrier. No effect on the Km or Vmax of Gly-Pro transport in this range was seen in the presence of the dipeptides Gly-Gly or cycloHis-Pro. The high-affinity carrier exhibited a different inhibition spectrum. Competitive inhibition of Gly-Pro transport was demonstrated for the dipeptides Gly-Gly and Gly-Sar. However, none of the other peptides tested above altered Gly-Pro transport in the high-affinity range, including pyroGlu-His, which is transported by a high-affinity carrier. At both low (4 x 10(-8) M) and high (4 x 10(-3) M) concentrations, uptake of Gly-Pro was stimulated in the presence of an inwardly directed H+ gradient but was unaffected by the presence of an inward Na+ gradient. In addition, measurements in the presence of valinomycin and an outwardly directed K+ gradient strongly suggest that H(+)-stimulated uptake at both concentrations is electrogenic.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals pH-dependent interconversion of two forms of tyrosinase in human skin

1988 ◽  
Vol 252 (2) ◽  
pp. 481-487 ◽  
Author(s):  
R K Tripathi ◽  
C Chaya Devi ◽  
A Ramaiah
Keyword(s):  
Human Skin ◽  
Sodium Phosphate ◽  
Inhibitory Concentration ◽  
Low Ph ◽  
The Other ◽  
Gradual Change ◽  
Ph Values ◽  
Sodium Phosphate Buffer ◽  
Solubilized Enzyme ◽  
Ph Dependent

1. We have shown that the characteristic lag in cresolase activity of human skin tyrosinase at inhibitory concentration of tyrosine was absent at all pH values studied, i.e. pH 5.2, 5.7, 6.2 and 6.8, if the enzyme solubilized at low pH was used as the source of enzyme, but the same enzyme when dialysed against buffers of various pH values showed linear activity only at pH 5.2 and was not inhibited by excess tyrosine, whereas at higher pH values it exhibited a lag and inhibition by excess tyrosine. 2. However, the enzyme solubilized in buffer/detergent, pH 6.8, when dialysed against buffer of the same pH showed linear activity at pH 5.2 and non-linear activity at pH 6.8. 3. The water/detergent-solubilized enzyme from human skin melanosomes showed linear activity even at inhibitory concentrations of tyrosine at pH 5.2 and 6.8 up to 2 h, but acceleration of rate was observed after 2 h for the enzyme measured at pH 6.8. 4. After dialysis of the water/detergent-solubilized enzyme against double-glass-distilled water, it still exhibits linear activity at inhibitory concentration of tyrosines at pH 6.8 for the first 2 h, but the same enzyme when dialysed against 0.02 M-sodium phosphate buffer, pH 6.8, exhibits negligible activity up to 1/2 h, in contrast with considerable activity before dialysis during the same interval of time, but without any loss of activity at later intervals of incubation time. 5. On the basis of these results, it is concluded that the enzyme exists in at least two interconvertible forms, one without lag and inhibition by excess tyrosine and the other with lag and inhibition by excess tyrosine. These two forms are interconvertible only by gradual change in pH over a period of hours.

scholarly journals The Properties of Cu Ions in Zeolites CuY Studied by IR Spectroscopy

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4686
Author(s):  
Jerzy Podobiński ◽  
Mariusz Gackowski ◽  
Grzegorz Mordarski ◽  
Katarzyna Samson ◽  
Michał Śliwa ◽  
...  
Keyword(s):  
Ir Spectroscopy ◽  
Positive Charge ◽  
Co Adsorption ◽  
The Other ◽  
Probe Molecules ◽  
Adsorption And Desorption ◽  
Other Hand ◽  
Close Proximity ◽  
No And Co Adsorption ◽  
Cu Ions

The properties of both Cu2+ and Cu+ ions in zeolite CuY were followed with NO and CO as probe molecules. Cu2+ was found to be located in SII, SII*, and SIII sites, whereas Cu+ was found in SII and SII* sites. The fine analysis of the spectra of Cu2+-NO and Cu+-CO adducts suggests that both in SII and in SII* sites two kinds of Cu cations exist. They differ in the positive charge, which may be related to the varying numbers of AlO4− in close proximity. The experiments of NO and CO adsorption and desorption evidenced that both Cu2+ and Cu+ sites of highest positive charge bind probe molecules most strongly but activate them to a lesser extent than the Cu sites of lowest positive charge. The experiments of reduction with hydrogen evidenced that the Cu ions of higher positive charge are first reduced by hydrogen. On the other hand, Cu sites of the lowest positive charge are first oxidized by oxygen. The experiments with CuNaY zeolites of various Cu contents suggest that the first introduced Cu (at low Cu contents) created Cu+, which was the most neutralized by framework oxygens. Such Cu cations are the most stabilized by framework oxygens.

scholarly journals Tripodal, Squaramide-Based Ion Pair Receptor for Effective Extraction of Sulfate Salt

Molecules ◽  
2021 ◽  
Vol 26 (9) ◽  
pp. 2751
Author(s):  
Damian Jagleniec ◽  
Marcin Wilczek ◽  
Jan Romański
Keyword(s):  
Ion Pairs ◽  
Binding Mode ◽  
Selective Extraction ◽  
Ion Pair ◽  
Hofmeister Series ◽  
Sulfate Salt ◽  
Lower Affinity ◽  
High Affinity ◽  
Binding Domains ◽  
Hydrated Sulfate

Combining three features—the high affinity of squaramides toward anions, cooperation in ion pair binding and preorganization of the binding domains in the tripodal platform—led to the effective receptor 2. The lack of at least one of these key elements in the structures of reference receptors 3 and 4 caused a lower affinity towards ion pairs. Receptor 2 was found to form an intramolecular network in wet chloroform, which changed into inorganic–organic associates after contact with ions and allowed salts to be extracted from an aqueous to an organic phase. The disparity in the binding mode of 2 with sulfates and with other monovalent anions led to the selective extraction of extremely hydrated sulfate anions in the presence of more lipophilic salts, thus overcoming the Hofmeister series.

Immunochemical Studies on Antithrombin III

1979 ◽  
Author(s):  
E.J. McKay
Keyword(s):  
Antithrombin Iii ◽  
The Other ◽  
Antigenic Determinants ◽  
Immunochemical Analysis ◽  
Paradoxical Effect ◽  
Test Protocol ◽  
Agar Gel ◽  
High Affinity ◽  
Time Required ◽  
Antigen Antibody

Depressed Antithrombin III (AT) levels Increase thrombic tendency in man, therefore value in assaying this protein has been established. Immunochemical analysis of AT in clinical disease has however proved controversial, consequently systematic studies were undertaken to rationalize the requirements necessary to optimise these methods in particular electro-Immunoassay. The known binding affinity of AT for heparin has been exploited to differentiate high affinity AT from its inhibitor - protease complexes and has resulted in reports stating that heparin added to the agar gel prior to electrophoresis significantly reduces the time required for completion of antigen/antibody complexes. Our studies however have demonstrated that the antibody required for quantitative analysis must be capable of not only reacting with “native” antigenic determinants of AT but also with “neo” antigens that are exposed when inhibitor-protease complexes are formed. Heparin should not be used in the test protocol, for it has a paradoxical effect on Immunopreclpltation in gels, masking some antigenic determinants of unbound - high affinity AT on one hand, and appear to disrupt the Immunoprecipitin “rocket” formed with the inhibitor-protease complexes during electrophoresis on the other.

scholarly journals Pollution Characteristics, Distribution and Ecological Risk of Potentially Toxic Elements in Soils from an Abandoned Coal Mine Area in Southwestern China

Minerals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 330
Author(s):  
Libo Pan ◽  
Xiao Guan ◽  
Bo Liu ◽  
Yanjun Chen ◽  
Ying Pei ◽  
...  
Keyword(s):  
Ecological Risk ◽  
Toxic Elements ◽  
Low Ph ◽  
Potentially Toxic Elements ◽  
Point Sources ◽  
The Other ◽  
Guizhou Province ◽  
Pollution Level ◽  
Agricultural Activity ◽  
Background Values

Acid mine drainage (AMD) from abandoned coal mines can lead to serious environmental problems due to its low pH and high concentrations of potentially toxic elements. In this study, soil pH, sulfur (S) content, and arsenic (As), cadmium (Cd), chromium (Cr), copper (Cu), lead (Pb), nickel (Ni), zinc (Zn), iron (Fe), manganese (Mn), and mercury (Hg) concentrations were measured in 27 surface soil samples from areas in which coal-mining activities ceased nine years previously in Youyu Catchment, Guizhou Province, China. The soil was acidic, with a mean pH of 5.28. Cadmium was the only element with a mean concentration higher than the national soil quality standard. As, Cd, Cu, Ni, Zn, Mn, Cr, and Fe concentrations were all higher than the background values in Guizhou Province. This was especially true for the Cd, Cu, and Fe concentrations, which were 1.69, 1.95, and 12.18 times their respective background values. The geoaccumulation index of Cd and Fe was present at unpolluted to moderately polluted and heavily polluted levels, respectively, indicating higher pollution levels than for the other elements in the study area. Spatially, significantly high Fe and S concentrations, as well as extremely low pH values, were found in the soils of the AMD sites; however, sites where tributaries merged with the Youyu River (TM) had the highest Cd pollution level. Iron originated mainly from non-point sources (e.g., AMD and coal gangues), while AMD and agricultural activity were the predominant sources of Cd. The results of an eco-risk assessment indicated that Cd levels presented a moderate potential ecological risk, while the other elements all posed a low risk. For the TM sites, the highest eco-risk was for Cd, with levels that could be harmful for aquatic organisms in the wet season, and may endanger human health via the food chain.

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