The bacterial Kdp K + -ATPase and its relation to other transport ATPases, such as the Na + /K + - and Ca 2+ -ATPases in higher organisms

1990 ◽  
Vol 326 (1236) ◽  
pp. 479-487 ◽  
Keyword(s):  
Turgor Pressure ◽  
The Other ◽  
Regulatory Proteins ◽  
Nodule Formation ◽  
Similar Function ◽  
Genetic Studies ◽  
High Affinity ◽  
Transmembrane Channel ◽  
Transport Atpases ◽  
Phosphate Regulon

The Kdp system is a three-subunit member of the Ej—E2 family of transport ATPases. There is sequence homology of the 72 kDa KdpB protein, the largest subunit of Kdp, with the other members of this family. The predicted structure of the 21 kDa KdpC subunit resembles that of the |3 subunit of the Na + ,K + -ATPase, suggesting that these subunits may have a similar function. The 59 kDa KdpA subunit has no known homologue; it is very hydrophobic and is predicted to cross the membrane 10-12 times. Genetic studies implicate this subunit in the binding of K + . As the binding site must be close to the beginning of the transmembrane channel, we suggest that KdpA also forms most or all of the latter. KdpA may have evolved from a K + /H + antiporter that was recruited by the KdpB precursor to achieve the high affinity and specificity for K + , and the activation of transport by low turgor pressure characteristic of Kdp. Turgor pressure controls the expression of Kdp. This action is dependent on the 70 kDa KdpD and 23 kDa KdpE proteins. We are in the process of sequencing these genes. KdpE is homologous to the smaller protein of other members of a family of pairs of regulatory proteins implicated in control of a variety of bacterial processes such as porin synthesis, phosphate regulon expression, nitrogen metabolism, chemotaxis and nodule formation.

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.

NaCl-dependent expression of amiloride-blockable Na+ channel in Xenopus oocytes

1992 ◽  
Vol 262 (2) ◽  
pp. G244-G248 ◽  
Author(s):  
C. Asher ◽  
D. Singer ◽  
R. Eren ◽  
O. Yeger ◽  
N. Dascal ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
The Other ◽  
Na Channel ◽  
Channel Activity ◽  
High Affinity ◽  
Other Hand ◽  
Exogenous Rna ◽  
Lower Intestine ◽  
Regulation Of Transport

RNA was isolated from chicken lower intestine (both colon and coprodeum) and injected into Xenopus oocytes. 22Na+ fluxes measured after 1-4 days demonstrated the induction of an amiloride-blockable pathway. The Na+ transporter expressed by the exogenous RNA had a high affinity to amiloride (inhibitory constant less than 0.1 microM), but was insensitive to ethylisopropyl amiloride, i.e., it is likely to be the apical Na+ channel. Functional channels were readily expressed in oocytes injected with RNA derived from chickens fed a low-NaCl diet. On the other hand, no channel activity was detected in oocytes injected with RNA isolated from chickens fed a high-NaCl diet. Thus the previously reported regulation of transport by the dietary NaCl intake involves modulations in the level of mRNA that codes either for the Na+ channel or a posttranscriptional regulator of the channel.

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 Genetics of HUS: the impact of MCP, CFH, and IF mutations on clinical presentation, response to treatment, and outcome

Blood ◽  
2006 ◽  
Vol 108 (4) ◽  
pp. 1267-1279 ◽  
Author(s):  
Jessica Caprioli ◽  
Marina Noris ◽  
Simona Brioschi ◽  
Gaia Pianetti ◽  
Federica Castelletti ◽  
...  
Keyword(s):  
Disease Recurrence ◽  
Factor H ◽  
Response To Therapy ◽  
Regulatory Proteins ◽  
Response To Treatment ◽  
Complement Factor ◽  
Genetic Studies ◽  
Complement Regulatory Proteins ◽  
Uremic Syndrome ◽  
The Impact

Abstract Hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy with manifestations of hemolytic anemia, thrombocytopenia, and renal impairment. Genetic studies have shown that mutations in complement regulatory proteins predispose to non–Shiga toxin–associated HUS (non-Stx–HUS). We undertook genetic analysis on membrane cofactor protein (MCP), complement factor H (CFH), and factor I (IF) in 156 patients with non-Stx–HUS. Fourteen, 11, and 5 new mutational events were found in MCP, CFH, and IF, respectively. Mutation frequencies were 12.8%, 30.1%, and 4.5% for MCP, CFH, and IF, respectively. MCP mutations resulted in either reduced protein expression or impaired C3b binding capability. MCP-mutated patients had a better prognosis than CFH-mutated and nonmutated patients. In MCP-mutated patients, plasma treatment did not impact the outcome significantly: remission was achieved in around 90% of both plasma-treated and plasma-untreated acute episodes. Kidney transplantation outcome was favorable in patients with MCP mutations, whereas the outcome was poor in patients with CFH and IF mutations due to disease recurrence. This study documents that the presentation, the response to therapy, and the outcome of the disease are influenced by the genotype. Hopefully this will translate into improved management and therapy of patients and will provide the way to design tailored treatments.

GRP-preferring bombesin receptors increase generation of inositol phosphates and tension in rat myometrium

1993 ◽  
Vol 265 (6) ◽  
pp. C1579-C1587 ◽  
Author(s):  
F. Amiot ◽  
D. Leiber ◽  
S. Marc ◽  
S. Harbon
Keyword(s):  
Methyl Ester ◽  
Inositol Phosphates ◽  
Regulatory Proteins ◽  
Single Class ◽  
Gastrin Releasing Peptide ◽  
Uterine Contractions ◽  
High Affinity ◽  
Neuromedin B ◽  
Guanine Nucleotide Regulatory Proteins ◽  
Relative Potencies

In the estrogen-treated rat myometrium, bombesin (Bn) and related agonists triggered contraction and the increased generation of inositol phosphates. The relative order of potencies was identical for both responses: Bn = gastrin releasing peptide (GRP) = litorin = neuromedin C >> neuromedin B. Two specific GRP-preferring receptor antagonists, namely [D-Phe6]Bn-(6-13) methyl ester and [Leu14,psi 13-14]Bn were inhibitory for both Bn-mediated tension and generation of inositol phosphates. [125I-Tyr4]Bn bound to myometrial membranes with high affinity (Kd = 104 pM) to a single class of sites in a saturable and reversible manner. The relative potencies for inhibiting binding were GRP = litorin = [Tyr4]Bn (Ki = 0.4 to 0.6 nM) >> neuromedin B (Ki = 10.3 nM). The high affinity displayed by [D-Phe6]Bn-(6-13) methyl ester (Ki = 2.8 nM) and [Leu14,psi 13-14]Bn (Ki = 35 nM) for competing for [Tyr4]Bn binding supported the involvement of a GRP-preferring Bn receptor. Guanine nucleotides decreased the binding of [125I-Tyr4]Bn and accelerated the rate of ligand dissociation, reflecting the coupling of receptors to guanine nucleotide regulatory proteins (G proteins). The results demonstrate that rat myometrium expresses functional GRP-preferring Bn receptors whose activation stimulates the phospholipase C pathway, pertussis toxin-insensitive event that contributes to Bn-mediated uterine contractions.

scholarly journals A novel method of sintering hybrid steels in an improved semiclosed container system

2013 ◽  
Vol 45 (3) ◽  
pp. 379-383 ◽  
Author(s):  
A. Cias
Keyword(s):  
Mechanical Properties ◽  
Structural Steels ◽  
Laboratory Scale ◽  
The Other ◽  
Reduction Reactions ◽  
High Affinity ◽  
Conventional Sintering ◽  
Superior Mechanical Properties ◽  
Novel Method

Conventional sintering techniques for structural steels have been developed principally for Cu and Ni containing alloys. Applying these to Cr and Mn steels (successful products of traditional metallurgy) encounter the problem of the high affinity for oxygen of these elements. A solution is employing a microatmosphere in a semiclosed container which favours reduction reactions. This has already proved successful on a laboratory scale, especially with nitrogen as the furnace gas. Further modifications to the system, now described, include the use of two sintering boxes, one inside the other. Superior mechanical properties, even using air as the furnace gas, are attainable.

THE INHERITANCE OF LEAF RUST RESISTANCE IN SEVEN VARIETIES OF COMMON WHEAT

1961 ◽  
Vol 41 (2) ◽  
pp. 342-359 ◽  
Author(s):  
R. G. Anderson
Keyword(s):  
Leaf Rust ◽  
Common Wheat ◽  
Rust Resistance ◽  
Leaf Rust Resistance ◽  
Susceptible Variety ◽  
The Other ◽  
Seedling Resistance ◽  
Genetic Studies ◽  
Type Reaction ◽  
Biological Studies

The inheritance of seedling resistance to races 1a, 5a, 11, 15a and 126a of leaf rust was studied in the varieties Exchange and Selkirk and to races 1a and 15a in the varieties Lee, Gabo, Timstein, Mayo 52 and Mayo 54. Thatcher was used as the susceptible variety. Rust tests were carried out on F1 and F2 populations of diallel crosses among these varieties and on F2 families from the backcrosses to Thatcher. Two genes were found. One gene LrE conditions a (2) type reaction to all five races in Exchange and Selkirk. The other gene LrL conditions a (; 1 =) type reaction to races 1a and 15a in all seven varieties. Isogenic lines possessing these genes are being developed in the varieties Prelude and Thatcher. The importance of such lines in future genetic studies and their application in other biological studies are discussed.The increase in amount of leaf rust found on Lee and Selkirk in Canada during the period 1951–1958 is accounted for by the increase of races which render the gene LrL ineffective in these two varieties.

Carbachol-induced down-regulation of high-affinity receptors for vasoactive intestinal peptide

1989 ◽  
Vol 257 (3) ◽  
pp. G402-G408
Author(s):  
M. Murakami ◽  
R. Vinayek ◽  
R. T. Jensen ◽  
J. D. Gardner
Keyword(s):  
Vasoactive Intestinal Peptide ◽  
Response Curve ◽  
Cholinergic Receptors ◽  
The Other ◽  
Tetraacetic Acid ◽  
Muscarinic Cholinergic Receptors ◽  
Pancreatic Acini ◽  
Vip Receptors ◽  
High Affinity ◽  
Muscarinic Cholinergic

When dispersed acini from guinea pig pancreas are first incubated with carbachol, the subsequent binding of 125I-vasoactive intestinal peptide (VIP) is inhibited during a second incubation. This inhibitory action of carbachol on binding of 125I-VIP depends on time, temperature, and the concentration of carbachol in the first incubation and can be blocked by atropine. First incubating acini with A23187, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), cholecystokinin octapeptide, bombesin, or 12-O-tetradecanoylphorbol-13-acetate does not alter binding of 125I-VIP. Adding EGTA to the first incubation medium abolishes the effect of carbachol on binding of 125I-VIP. In control acini or acini first incubated with carbachol, approximately half of the bound 125I-VIP can be stripped by acetic acid. 125I-VIP interacts with two distinct classes of receptors on pancreatic acini. One has a high affinity for VIP (Kd, 1 nM); the other has a low affinity for VIP (Kd, 2 microM). First incubating acini with carbachol decreases the number but not the affinity of high-affinity VIP receptors with no change in the number or affinity of low-affinity VIP receptors. Pancreatic acini possess two classes of muscarinic cholinergic receptors: one has a high affinity (Kd, 4 microM) and the other has a low affinity (Kd, 698 microM) for carbachol. The dose-response curve for carbachol-induced inhibition of binding of 125I-VIP and that for occupation of low-affinity muscarinic cholinergic receptors by carbachol are similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of bombesin receptors on pancreatic acini by cholecystokinin

1989 ◽  
Vol 256 (2) ◽  
pp. G291-G298
Author(s):  
M. Younes ◽  
S. A. Wank ◽  
R. Vinayek ◽  
R. T. Jensen ◽  
J. D. Gardner
Keyword(s):  
Guinea Pig ◽  
Response Curve ◽  
Binding Capacity ◽  
The Other ◽  
Single Class ◽  
Pancreatic Acini ◽  
Cck Receptors ◽  
Cyclic Monophosphate ◽  
High Affinity ◽  
Bombesin Receptors

When guinea pig pancreatic acini are first incubated with the COOH-terminal octapeptide of cholecystokinin (CCK-8), washed, and then reincubated with 125I-[Tyr4]bombesin (125I-[Tyr4]BN) there is a significant decrease in binding of 125I-[Tyr4]BN compared with that observed with pancreatic acini that have been first incubated with no additions. The CCK-8-induced decrease in binding is maximal after 90 min of first incubation is abolished by reducing the temperature of the first incubation from 37 to 4 degrees C or by adding L364,718 to the first incubation and cannot be reproduced by first incubating acini with A23187, 8-bromoadenosine 3',5'-cyclic monophosphate (8Br-cAMP), 8-bromoadenosine 3',5'-cyclic monophosphate (8Br-cGMP), or 12-O-tetradecanoylphorbol 13-acetate. 125I-[Tyr4]BN interacts with a single class of receptors on pancreatic acini, and first incubating acini with CCK-8 decreases the affinity of BN receptors for BN with no change in the maximal binding capacity. CCK-8 does not alter the rate at which bound 125I-[Tyr4]BN dissociates from pancreatic acini; therefore, CCK-8 must alter the rate at which the radiolabeled BN analogue associates with its receptor. Pancreatic acini possess two classes of CCK receptors: one has a high affinity for CCK-8; the other has a low affinity for CCK-8. The dose-response curve for CCK-8-induced inhibition of binding of 125I-[Tyr4]BN appears to to reflect occupation of low-affinity CCK receptors by CCK-8.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Genetic Aspects of Sickle Cell Anemia and Microdrepanocytic Disease

Blood ◽  
1952 ◽  
Vol 7 (4) ◽  
pp. 429-435 ◽  
Author(s):  
E. SILVESTRONI ◽  
I. BIANCO
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Sickle Cell Trait ◽  
Genetic Data ◽  
The Other ◽  
Moderate Degree ◽  
Simultaneous Presence ◽  
Genetic Studies ◽  
Asymptomatic Individuals ◽  
Degree Of Severity

Abstract 1. A brief review is presented of the genetic theories of sickle cell anemia and the sickle cell trait. 2. The genetic data on 2 families of asymptomatic individuals with the sickle cell trait and of 3 families of patients with sickle cell anemia are reported. These data confirm the heterozygous-homozygous theory of Neel. 3. The possibility is considered that many of the cases of sickle cell anemia described in the white race are actually examples of "microdrepanocytic disease." 4. Microdrepanocytic disease is a new syndrome, first described by the authors from Italy. It has some of the characteristics of both sickle cell anemia and Mediterranean anemia. On the basis of studies in 11 families, the presence of the sickle cell trait in one parent and of microcythemia (Mediterranean anemia trait) in the other, results in microdrepanocytic disease in some of the offspring. Hematologic studies in these patients indicate the simultaneous presence of both sickle cell and microcythemic genes. 5. Genetic studies of these families suggests that the genes for microcythemia and for sicklemia are located on different chromosomes and are inherited independently of each other. On the other hand, their simultaneous presence leads to a disease of a moderate degree of severity having many of the features of sickle cell anemia.

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