scholarly journals Purification and some properties of cytosolic cobalamin-binding protein in Euglena gracilis

1987 ◽  
Vol 247 (3) ◽  
pp. 679-685 ◽  
Author(s):  
F Watanabe ◽  
Y Nakano ◽  
S Kitaoka
Keyword(s):  
Euglena Gracilis ◽  
Binding Proteins ◽  
Microsomal Fraction ◽  
Binding Protein ◽  
Physiological Role ◽  
Binding Activity ◽  
Thiol Groups ◽  
Double Immunodiffusion ◽  
Ph Dependency ◽  
Ks Values

In Euglena gracilis SM-ZK, a bleached mutant of E. gracilis z, the cobalamin- (Cbl-)binding activity was distributed in cytosol (49.2%), mitochondria (20.3%) and microsomal fraction (20.4%). The cytosolic Cbl-binding activity gave two major peaks in isoelectric focusing. The Cbl-binding protein with pI 3.2 was purified 6500-fold in a yield of 19.9%, and that with pI 4.7 5800-fold in a yield of 11.9%. The monomeric Mr values of both Cbl-binding proteins were about 66,000. The Cbl-binding activity of both proteins showed a very low pH-dependency, and thiol groups and metal ions were not concerned with the Cbl-binding activities. The Ks values of the Cbl-binding proteins with pI 3.8 and 4.7 for CN-Cbl were 1.0 and 2.0 nM respectively. The Cbl-binding protein with pI 3.8 was shown to be immunologically identical with the protein with pI 4.7 by double-immunodiffusion experiments against antibody to the protein with pI 3.8. The two cytosolic Cbl-binding proteins did not show the activities of Cbl-dependent enzymes in E. gracilis, N5-methyltetrahydrofolate:homocysteine methyltransferase, methylmalonyl-CoA mutase and ribonucleotide reductase, suggesting that the two cytosolic Cbl-binding proteins play a physiological role as intracellular Cbl carriers.

scholarly journals A yeast protein that binds nuclear localization signals: purification localization, and antibody inhibition of binding activity.

1991 ◽  
Vol 113 (6) ◽  
pp. 1243-1254 ◽  
Author(s):  
U Stochaj ◽  
M Osborne ◽  
T Kurihara ◽  
P Silver
Keyword(s):  
Nuclear Localization ◽  
Binding Proteins ◽  
Protein Import ◽  
Nuclear Protein ◽  
Binding Protein ◽  
Protein Localization ◽  
Binding Activity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Nuclear Localization Signals

Short stretches of amino acids, termed nuclear localization sequences (NLS), can mediate assembly of proteins into the nucleus. Proteins from the yeast, Saccharomyces cerevisiae, have been identified that specifically recognize nuclear localization peptides (Silver, P., I. Sadler, and M. A. Osborne. 1989. J. Cell Biol. 109:983-989). We now further define the role of one of these NLS-binding proteins in nuclear protein localization. The NLS-binding protein of 70-kD molecular mass can be purified from salt extracts of nuclei. Antibodies raised against the NLS-binding protein localized the protein mainly to the nucleus with minor amounts in the cytoplasm. These antibodies also inhibited the association of NLS-protein conjugates with nuclei. Incubation of nuclei with proteases coupled to agarose removed NLS-binding protein activity. Extracts enriched for NLS-binding proteins can be added back to salt or protease-treated nuclei to restore NLS-binding activity. These results suggest that the first step of nuclear protein import can be reconstituted in vitro.

Comparisons of various acidic treatments of bovine serum on insulin-like growth factor-I immunoreactivity and binding activity

1990 ◽  
Vol 127 (1) ◽  
pp. 139-148 ◽  
Author(s):  
C. Y. Lee ◽  
D. M. Henricks
Keyword(s):  
Growth Factor ◽  
Bovine Serum ◽  
Binding Proteins ◽  
Binding Protein ◽  
Gel Filtration ◽  
Binding Activity ◽  
Insulin Like Growth Factor ◽  
Ethanol Extraction ◽  
Factor I ◽  
Igf I

ABSTRACT Untreated serum exhibited two forms of insulin-like growth factor-I (IGF-I)-binding protein complexes during gel chromatography: one of Mr 150 000 and the other of Mr 40 000–45 000. The majority of the immunoreactive IGF-I was associated with the Mr 150 000 complex. Following acid-ethanol extraction of serum, the binding activity at Mr 150 000 disappeared and a reduced binding activity appeared in the albumin size range. Acid incubation of serum was slightly less effective than acid-ethanol extraction in reducing the binding activity. Acid-ethanol-extracted or acid-incubated serum were parallel to IGF-I standard in the dose–response displacement of iodinated IGF-I. Gel filtration of serum with 1 mol acetic acid/l almost completely separated IGF-I and the binding proteins. Binding-protein fractions from gel filtration interfered with the immunoreactivity of IGF-I with its antibodies, causing a non-parallel displacement curve in the radioimmunoassay (RIA). Serum IGF-I could be isolated as a single peak by high performance C18 reverse-phase liquid chromatography (HPLC). The concentrations of IGF-I measured in bovine sera by RIA were similar between acid gel filtration and HPLC; the concentrations by acid-ethanol extraction and acid incubation being about 30% smaller than those measured with former methods. The lower concentration of IGF-I measured in bovine serum with acid-ethanol extraction or acid incubation appears to be due to interference of IGF-binding proteins not removed by either treatment. Journal of Endocrinology (1990) 127, 139–148

scholarly journals Calcium-ion-binding activity in human small-intestinal mucosal cytosol Purification of two proteins and interrelationship of calcium-binding fractions

1981 ◽  
Vol 197 (1) ◽  
pp. 55-65 ◽  
Author(s):  
M Davie
Keyword(s):  
Molecular Weight ◽  
Calcium Binding ◽  
Binding Proteins ◽  
Binding Protein ◽  
Mammalian Species ◽  
Binding Activity ◽  
Calcium Binding Proteins ◽  
Calcium Binding Protein ◽  
Small Intestinal Mucosa ◽  
Small Intestinal

Ca2+-binding activity was investigated in human small-intestinal mucosal cytosol. Binding was detected in fractions with molecular weights of 28000 and about 900000, as determined by gel filtration. No binding was found at molecular weight 12000-13000 (the molecular weight of calcium-binding protein in lower mammalian species) until the cytosol had been subjected to a hollow-fibre-filtration step. The appearance of Ca2+-binding at molecular weight 12000-13000 was associated with a decline in the 28000-mol.wt. calcium-binding fraction. The 12000-13000-mol.wt. fraction contained two distinct calcium-binding proteins. One of these proteins had properties similar to those of pig calcium-binding protein. Antiserum to this protein reacted against the 28000-mol.wt calcium-binding fraction in cytosol from human small-intestinal mucosa and from human kidney. An immunoassay method for one of the calcium-binding proteins was established. In normal duodenal mucosa the concentration was 915 micrograms/g and in the ileum it was 443 micrograms/g of mucosa. A subject with hypercalcaemic sarcoidosis had 1200 micrograms/g of mucosa in the jejunum, and a subject with an undetectable concentration of plasma 25-hydroxycholecalciferol had concentrations of calcium-binding protein in the mucosa similar to those found in normal subjects.

scholarly journals Molecular evidence of intertidal habitats selecting for repeated ice-binding protein evolution in invertebrates

2021 ◽  
Author(s):  
Isaiah C. H. Box ◽  
Benjamin J. Matthews ◽  
Katie E. Marshall
Keyword(s):  
Binding Proteins ◽  
Binding Protein ◽  
Antifreeze Proteins ◽  
Binding Activity ◽  
Ecological Groups ◽  
Type Ii ◽  
Antifreeze Glycoproteins ◽  
Ice Binding ◽  
Ice Binding Protein ◽  
Intertidal Invertebrates

ABSTRACTIce-binding proteins (IBPs) have evolved independently in multiple taxonomic groups to improve their survival of sub-zero temperatures. Intertidal invertebrates in temperate and polar regions frequently encounter sub-zero temperatures, yet there is little information on IBPs in these organisms. We hypothesized that there are far more ice-binding proteins than are currently known and that the occurrence of freezing in the intertidal zone selects for these proteins. We compiled a list of genome-sequenced invertebrates across multiple habitats and a list of known IBP sequences and used BLAST to identify a wide array of putative IBPs in those invertebrates. We found that the probability of an invertebrate species having an ice-binding protein was significantly greater in intertidal species as compared to those primarily found in open ocean or freshwater habitats. These intertidal IBPs had high sequence similarity to fish and tick antifreeze glycoproteins and fish type II antifreeze proteins. Previously established classifiers based on machine learning techniques further predicted ice-binding activity in the majority of our newly identified putative IBPs. We investigated the potential evolutionary origin of one putative IBP from the hard-shelled mussel Mytilus coruscus and suggest that it arose through gene duplication and neofunctionalization. We show that IBPs likely readily evolve in response to freezing risk, that there is an array of uncharacterized ice binding proteins and highlight the need for broader laboratory-based surveys of the diversity of ice binding activity across diverse taxonomic and ecological groups.Summary statementIntertidal invertebrates have a disproportionate number of putative ice-binding proteins relative to other habitats. These putative proteins are highly similar to antifreeze glycoproteins and type II antifreeze proteins from fish.

Small GTP-binding Protein RalA Associates with Weibel-Palade Bodies in Endothelial Cells

1999 ◽  
Vol 82 (09) ◽  
pp. 1177-1181 ◽  
Author(s):  
Hubert de Leeuw ◽  
Pauline Wijers-Koster ◽  
Jan van Mourik ◽  
Jan Voorberg
Keyword(s):  
Endothelial Cells ◽  
Binding Proteins ◽  
Binding Protein ◽  
Control Release ◽  
Small Gtpase ◽  
Gtp Binding Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
Gtp Binding Proteins ◽  
Dense Granules ◽  
Gtp Binding

SummaryIn endothelial cells von Willebrand factor (vWF) and P-selectin are stored in dense granules, so-called Weibel-Palade bodies. Upon stimulation of endothelial cells with a variety of agents including thrombin, these organelles fuse with the plasma membrane and release their content. Small GTP-binding proteins have been shown to control release from intracellular storage pools in a number of cells. In this study we have investigated whether small GTP-binding proteins are associated with Weibel-Palade bodies. We isolated Weibel-Palade bodies by centrifugation on two consecutive density gradients of Percoll. The dense fraction in which these subcellular organelles were highly enriched, was analysed by SDS-PAGE followed by GTP overlay. A distinct band with an apparent molecular weight of 28,000 was observed. Two-dimensional gel electrophoresis followed by GTP overlay revealed the presence of a single small GTP-binding protein with an isoelectric point of 7.1. A monoclonal antibody directed against RalA showed reactivity with the small GTP-binding protein present in subcellular fractions that contain Weibel-Palade bodies. The small GTPase RalA was previously identified on dense granules of platelets and on synaptic vesicles in nerve terminals. Our observations suggest that RalA serves a role in regulated exocytosis of Weibel-Palade bodies in endothelial cells.

Rap1b Association with the Platelet Cytoskeleton Occurs in the Absence of Glycoproteins IIb/IIIa

1998 ◽  
Vol 79 (04) ◽  
pp. 832-836 ◽  
Author(s):  
Thomas Fischer ◽  
Christina Duffy ◽  
Gilbert White
Keyword(s):  
Molecular Weight ◽  
Divalent Cation ◽  
Binding Proteins ◽  
Binding Protein ◽  
Platelet Membrane ◽  
Low Molecular Weight ◽  
Membrane Glycoproteins ◽  
Gtp Binding Protein ◽  
Gtp Binding Proteins ◽  
Gtp Binding

SummaryPlatelet membrane glycoproteins (GP) IIb/IIIa and rap1b, a 21 kDa GTP binding protein, associate with the triton-insoluble, activation-dependent platelet cytoskeleton with similar rates and divalent cation requirement. To examine the possibility that GPIIb/IIIa was required for rap1b association with the cytoskeleton, experiments were performed to determine if the two proteins were linked under various conditions. Chromatography of lysates from resting platelets on Sephacryl S-300 showed that GPIIb/IIIa and rap1b were well separated and distinct proteins. Immunoprecipitation of GPIIb/IIIa from lysates of resting platelets did not produce rap1b or other low molecular weight GTP binding proteins and immunoprecipitation of rap1b from lysates of resting platelets did not produce GPIIb/IIIa. Finally, rap1b was associated with the activation-dependent cytoskeleton of platelets from a patient with Glanzmann’s thrombasthenia who lacks surface expressed glycoproteins IIb and IIIa. Based on these findings, we conclude that no association between GPIIb/IIIa and rap1b is found in resting platelets and that rap1b association with the activation-dependent cytoskeleton is at least partly independent of GPIIb/IIIa.

Diagnostic value of fatty acid-binding protein determination in urologists and nephrologists clinical practice

2020 ◽  
pp. 92-99
Author(s):  
Konstantin R. Galkovich
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Binding Protein ◽  
Binding Protein ◽  
Physiological Role ◽  
Diagnostic Value ◽  
Obstructive Nephropathy ◽  
Malignant Neoplasms ◽  
Early Screening ◽  
Fatty Acid Binding ◽  
Acid Binding Protein

This review summarizes the data on the diagnostic value of determining the fatty acid-binding protein (FABP) in urological and nephrological diseases. A physiological role of this protein in the pathogenesis of malignant neoplasms of the kidney, bladder, and prostate was analyzed. The dynamics of FABP in serum and urine with decreased renal function was studied: this protein is considered as a diagnostic and prognostic marker for chronic kidney disease and acute renal injury. The value of FABP for early screening of patients with obstructive nephropathy was revealed, and its role in predicting the restoration of kidney function was studied: the dynamics of FABP content can characterize the process of graft recovery, determine the need for hemodialysis. In patients with oligozooastenospermia, a reduced content of FABP in the ejaculate was registered, which was probably an adverse sign indicating a violation of male fertility.

scholarly journals Biochemical and Initial Structural Characterization of the Monocot Chimeric Jacalin OsJAC1

2021 ◽  
Vol 22 (11) ◽  
pp. 5639
Author(s):  
Nikolai Huwa ◽  
Oliver H. Weiergräber ◽  
Christian Kirsch ◽  
Ulrich Schaffrath ◽  
Thomas Classen
Keyword(s):  
Physiological Role ◽  
Basic Function ◽  
Binding Activity ◽  
Carbohydrate Binding ◽  
Lectin Domain ◽  
Reducing Conditions ◽  
Transition Points ◽  
The Individual ◽  
High Yields ◽  
Dirigent Domain

The monocot chimeric jacalin OsJAC1 from Oryza sativa consists of a dirigent and a jacalin-related lectin domain. The corresponding gene is expressed in response to different abiotic and biotic stimuli. However, there is a lack of knowledge about the basic function of the individual domains and their contribution to the physiological role of the entire protein. In this study, we have established a heterologous expression in Escherichia coli with high yields for the full-length protein OsJAC1 as well as its individual domains. Our findings showed that the secondary structure of both domains is dominated by β-strand elements. Under reducing conditions, the native protein displayed clearly visible transition points of thermal unfolding at 59 and 85 °C, which could be attributed to the lectin and the dirigent domain, respectively. Our study identified a single carbohydrate-binding site for each domain with different specificities towards mannose and glucose (jacalin domain), and galactose moieties (dirigent domain), respectively. The recognition of different carbohydrates might explain the ability of OsJAC1 to respond to different abiotic and biotic factors. This is the first report of specific carbohydrate-binding activity of a DIR domain, shedding new light on its function in the context of this monocot chimeric jacalin.

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