scholarly journals Identification of membrane-bound CR1 (CD35) in human urine: evidence for its release by glomerular podocytes.

1994 ◽  
Vol 179 (3) ◽  
pp. 889-899 ◽  
Author(s):  
M Pascual ◽  
G Steiger ◽  
S Sadallah ◽  
J P Paccaud ◽  
J L Carpentier ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Lupus Erythematosus ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Apparent Size ◽  
Soluble Form ◽  
Focal Sclerosis ◽  
Membrane Bound ◽  
The Difference ◽  
Normal Urine

Complement receptor 1 (CR1) is present on erythrocytes (E-CR1), various leucocytes, and renal glomerular epithelial cells (podocytes). In addition, plasma contains a soluble form of CR1 (sCR1). By using a specific ELISA, CR1 was detected in the urine (uCR1) of normal individuals (excretion rate in 12 subjects, 3.12 +/- 1.15 micrograms/24 h). Contrary to sCR1, uCR1 was pelleted by centrifugation at 200,000 g for 60 min. Analysis by sucrose density gradient ultracentrifugation showed that uCR1 was sedimenting in fractions larger than 19 S, whereas sCR1 was found as expected in fractions smaller than 19 S. The addition of detergents reduced the apparent size of uCR1 to that of sCR1. After gel filtration on Sephacryl-300 of normal urine, the fractions containing uCR1 were found to be enriched in cholesterol and phospholipids. The membrane-association of uCR1 was demonstrated by analyzing immunoaffinity purified uCR1 by electron microscopy which revealed membrane-bound vesicles. The apparent molecular mass of uCR1 was 15 kD larger than E-CR1 and sCR1 when assessed by SDS-PAGE and immunoblotting. This difference in size could not be explained on the basis of glycosylation only, since pretreatment with N-glycosidase F reduced the size of all forms of CR1; however, the difference in regular molecular mass was not abrogated. The structural alleles described for E-CR1 were also found for uCR1. The urine of patients who had undergone renal transplantation contained alleles of uCR1 which were discordant with E-CR1 in 7 of 11 individuals, indicating that uCR1 originated from the kidney. uCR1 was shown to bind C3b-coated immune complexes, suggesting that the function of CR1 was not destroyed in urine. A decrease in uCR1 excretion was observed in 3 of 10 patients with systemic lupus erythematosus, corresponding to the three who had severe proliferative nephritis, and in three of three patients with focal sclerosis, but not in six other patients with proteinuria. Taken together, these data suggest that glomerular podocytes release CR1-coated vesicles into the urine. The function of this release remains to be defined, but it may be used as a marker for podocyte injury.

scholarly journals Effect of Fertilizer Application Level on Pectin Composition of Hakuho Peach (Prunus persica Batsch) During Maturation

HortScience ◽  
2006 ◽  
Vol 41 (7) ◽  
pp. 1571-1575 ◽  
Author(s):  
H.J. Jia ◽  
K. Mizuguchi ◽  
K. Hirano ◽  
G. Okamoto
Keyword(s):  
Molecular Mass ◽  
Prunus Persica ◽  
Gel Filtration ◽  
Fertilizer Application ◽  
Water Soluble ◽  
Mature Stage ◽  
Peach Fruit ◽  
The Difference ◽  
Ripe Stage ◽  
Early Degradation

Effects of fertilizer application levels on fruit texture and flesh pectin compositions of a melting peach were investigated. Hakuho trees (Prunus persica Batsch) were supplied with normal (M), high (H; M × 2), and superhigh (SH; M × 4) levels of complete liquid fertilizer twice a week. Flesh firmness of the H and SH treatment fruit was lower than that of M treatment fruit at the hard-mature and firm-mature stages, although no difference was detected at the full ripe stage. Sensory scores for flesh texture at the full ripe stage were highest in the N treatment fruit and lowest in the SH treatment fruit. The content of water-soluble polyuronides (WSP) in flesh was highest in SH fruit and lowest in M fruit at the hard-mature stage, although the difference became smaller at the full ripe stage. Molecular mass analysis using a gel filtration column revealed that water-soluble polysaccharides in alcohol-insoluble solids (AIS) of the H and SH fruits had a peak of high molecular mass, ≈200 kDa, at the hard-mature stage, and the molecular mass decreased gradually to ≈23 kDa at the full ripe stage. In the M fruit, however, the molecular mass was rather constant during the ripening period, 112 kDa even at the full ripe stage. The analysis of acidic fractions (pectin) in the polysaccharides using an ion exchange column, as well as juice gellation test by adding Ca and Tris buffer, also indicated that high levels of fertilizer application impairs an early degradation of flesh polyuronides resulting in the accumulation of low-molecular-weight WSP. This may ultimately cause the inferior flesh texture of overfertilized peach fruit.

Solubilization, characterization, and detergent interactions of lymphocyte 5′-nucleotidase

1989 ◽  
Vol 67 (4-5) ◽  
pp. 214-223 ◽  
Author(s):  
D. W. Loe ◽  
J. R. Glover ◽  
S. Head ◽  
F. J. Sharom
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Heat Stability ◽  
Sucrose Density Gradient ◽  
Sds Page ◽  
Molecular Masses ◽  
Break Points ◽  
Solubilized Enzyme ◽  
Single Subunit ◽  
Sucrose Density Gradient Sedimentation

5′-Nucleotidase is a member of a recently identified class of membrane proteins that is anchored via a phosphatidylinositol-containing glycolipid. The enzyme was readily solubilized with full retention of catalytic activity by nonionic and anionic detergents such as alkylthioglucosides, deoxycholate, and 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS), while the cationic detergent dodecyltrimethylammonium bromide (DTAB) caused loss of activity. 5′-Nucleotidase was released only at high detergent concentrations, suggesting that it is tightly associated with the membrane. DTAB and deoxycholate caused a loss of heat stability, while alkylthioglucosides had no effect. CHAPS produced a remarkable increase in the heat stability of the partially purified (glycoprotein fraction) and purified enzyme. Arrhenius plots of solubilized 5′-nucleotidase showed "break points" for all detergents in the temperature range 30–37 °C. SDS-PAGE of pure 5′-nucleotidase showed a single subunit of molecular mass 70 kilodaltons (kDa), while sucrose density gradient sedimentation gave a peak of activity corresponding to 132 kDa, indicating that the enzyme exists as a dimer. Gel filtration of the solubilized enzyme in several detergents showed apparent molecular masses between 200–630 kDa, suggesting that lymphocyte 5′-nucleotidase may be present in high molecular mass aggregates in its native state.Key words: 5′-nucleotidase, plasma membrane, detergents, solubilization, stability, activation energy.

scholarly journals Purification and characterization of rat epididymal-fluid α-d-mannosidase: similarities to sperm plasma-membrane α-d-mannosidase

1993 ◽  
Vol 290 (2) ◽  
pp. 427-436 ◽  
Author(s):  
D R P Tulsiani ◽  
M D Skudlarek ◽  
S K Nagdas ◽  
M C Orgebin-Crist
Keyword(s):  
Molecular Mass ◽  
Plasma Membranes ◽  
Antigenic Site ◽  
Protein Band ◽  
Soluble Form ◽  
Major Protein ◽  
Major Protein Band ◽  
Membrane Bound ◽  
Epididymal Fluid

We have previously reported the occurrence and partial characterization of a novel alpha-D-mannosidase activity on rat sperm plasma membranes [Tulsiani, Skudlarek and Orgebin-Crist (1989) J. Cell Biol. 109, 1257-1267]. Here, we report the presence of a similar alpha-D-mannosidase activity in a soluble form in rat epididymal fluid. The soluble enzyme was purified nearly 500-fold with 9-12% recovery to a state approaching homogeneity using: (1) (NH4)2SO4 precipitation; (2) affinity chromatography on immobilized mannan and D-mannosamine; (3) ion-exchange (DE-52) column chromatography; (4) molecular-sieve chromatography. The enzyme was eluted from the final column (Sephacryl S-400) at an apparent molecular mass of 460 kDa. When resolved by SDS/PAGE (under denaturing conditions), the enzyme showed a major protein band (115 kDa) and few very minor bands. The polyclonal antibody raised against the major protein band was found to cross-react with the alpha-D-mannosidase activity present in epididymal fluid (soluble) and detergent-solubilized spermatozoa from the rat and mouse. This result suggested that the soluble and membrane-bound enzyme activities shared a common antigenic site(s). The antibody was used to characterize further the alpha-D-mannosidase activity(ies) present in the rat epididymal fluid and rat sperm plasma membranes. Data from these studies show that the two forms are similar in (a) subunit molecular mass, (b) substrate specificity and (c) inhibitory effect of several sugars. These similarities suggest that the soluble and membrane-bound alpha-D-mannosidase activities are isoforms. Immunoprecipitation studies after solubilization of the testis and epididymal particulate fraction from sexually immature rats show that the testis (but not the epididymis) contains the immunoreactive alpha-D-mannosidase activity. This result and the fact that spermatozoa from the rat rete testis show alpha-D-mannosidase activity indicate that the sperm enzyme is synthesized in the testis during spermatogenesis.

scholarly journals Subunit structure of vacuolar proton-pyrophosphatase as determined by radiation inactivation

1996 ◽  
Vol 316 (1) ◽  
pp. 143-147 ◽  
Author(s):  
Chi M. TZENG ◽  
Chih Y. YANG ◽  
Su J. YANG ◽  
Shih S. JIANG ◽  
Soong Y. KUO ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Proton Translocation ◽  
Gel Filtration ◽  
Size Exclusion ◽  
Size Analysis ◽  
Subunit Structure ◽  
Radiation Inactivation ◽  
Membrane Bound ◽  
Solubilized Form ◽  
Functional Size

Vacuolar proton-pyrophosphatase (H+-PPase) of mung bean seedlings contains a single kind of polypeptide with a molecular mass of approx. 73 kDa. However, in this study, a molecular mass of approx. 140 kDa was obtained for the purified vacuolar H+-PPase by size-exclusion gel-filtration chromatography, suggesting that the solubilized form of this enzyme is a dimer. Radiation inactivation analysis of tonoplast vesicles yielded functional masses of 141.5±10.8 and 158.4±19.5 kDa for PPi hydrolysis activity and its supported proton translocation respectively. These results confirmed the in situ dimeric structure of the membrane-bound H+-PPase of plant vacuoles. Further target-size analysis showed that the functional unit of purified vacuolar H+-PPase was 71.1±6.7 kDa, indicating that only one subunit of the purified dimeric complex would sufficiently display its enzymic reaction. Moreover, in the presence of valinomycin and KCl, the functional size of membrane-bound H+-PPase was decreased to approx. 63.4±6.3 kDa. A working model was proposed to elucidate the structure of native H+-PPase on vacuolar membrane as a functional dimer. Factors that would disturb the membrane, e.g. membrane solubilization and the addition of valinomycin and KCl, may induce an alteration in its enzyme structure, subsequently resulting in a different functional size.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

1977 ◽  
Vol 37 (01) ◽  
pp. 073-080 ◽  
Author(s):  
Knut Gjesdal ◽  
Duncan S. Pepper
Keyword(s):  
Macaca Mulatta ◽  
Half Life ◽  
Human Platelet ◽  
Gel Filtration ◽  
Platelet Factor 4 ◽  
Active Protein ◽  
Platelet Factor ◽  
Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

scholarly journals Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA

1997 ◽  
Vol 325 (3) ◽  
pp. 761-769 ◽  
Author(s):  
Isabelle GARCIA ◽  
Matthew RODGERS ◽  
Catherine LENNE ◽  
Anne ROLLAND ◽  
Alain SAILLAND ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Gel Filtration ◽  
Peptide Sequence ◽  
Amino Acid Residues ◽  
Carrot Cells ◽  
Expression Studies ◽  
Sds Page ◽  
Molecular Masses ◽  
The Difference ◽  
Dioxygenase Activity

p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate. In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones. Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides. In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells. Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol. Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 45–46 kDa. This protein specifically cross-reacts with an antiserum raised against the p-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens. Gel-filtration chromatography indicates that the enzyme behaves as a homodimer. We also report the isolation and nucleotide sequence of a cDNA encoding a carrot p-hydroxyphenylpyruvate dioxygenase. The nucleotide sequence (1684 bp) encodes a protein of 442 amino acid residues with a molecular mass of 48094 Da and shows specific C-terminal regions of similarity with other p-hydroxyphenylpyruvate dioxygenases. This cDNA encodes a functional p-hydroxyphenylpyruvate dioxygenase, as evidenced by expression studies with transformed Escherichia coli cells. Comparison of the N-terminal sequence of the 45–46 kDa polypeptide purified from carrot cells with the deduced peptide sequence of the cDNA confirms that this polypeptide supports p-hydroxyphenylpyruvate dioxygenase activity. Immunodetection studies of the native enzyme in carrot cellular extracts reveal that N-terminal proteolysis occurs during the process of purification. This proteolysis explains the difference in molecular masses between the purified protein and the deduced polypeptide.

The Relation between the Concentration of Calcium Salts in the Urine and Renal Stone Composition in Patients with Calcium-Containing Renal Stones

1972 ◽  
Vol 43 (3) ◽  
pp. 433-441 ◽  
Author(s):  
R. W. Marshall ◽  
M. Cochran ◽  
W. G. Robertson ◽  
A. Hodgkinson ◽  
B. E. C. Nordin
Keyword(s):  
Calcium Phosphate ◽  
Calcium Oxalate ◽  
Renal Stones ◽  
Urine Ph ◽  
Stone Composition ◽  
Calcium Oxalate Stones ◽  
Urine Oxalate ◽  
The Difference ◽  
Normal Urine ◽  
Normal Men

1. Diurnal variations in urine calcium oxalate and calcium phosphate activity products were observed in normal men and patients with recurrent calcium oxalate or mixed oxalate—phosphate renal stones. 2. Maximum and minimum calcium oxalate products were higher in the patients than in the controls, the difference being most marked in the patients with calcium oxalate stones. 3. Maximum and minimum calcium phosphate products expressed as octocalcium phosphate [(Ca8H2(PO4)6], brushite or hydroxyapatite, were significantly higher than normal in the patients with mixed stones but not in the patients with calcium oxalate stones. 4. The raised calcium oxalate products in the patients were due mainly to increased concentrations of Ca2+ ions; these, in turn, were due mainly to an increased rate of excretion of calcium. Raised calcium phosphate products were due mainly to hypercalciuria, combined with abnormally high urine pH values. 5. Patients with recurrent calcium stones appear to fall into two types: (1) patients with calcium oxalate stones associated with hypercalciuria, a normal or raised urine oxalate and a normal urine pH; (2) patients with mixed oxalate—phosphate stones associated with hypercalciuria, a normal or raised urine oxalate and a raised urine pH. 6. The implications of these findings in regard to treatment are discussed.

scholarly journals Identification and characterization of Sulfolobus solfataricusD-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway

2005 ◽  
Vol 387 (1) ◽  
pp. 271-280 ◽  
Author(s):  
Seonghun KIM ◽  
Sun Bok LEE
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Genome Database ◽  
Sds Page ◽  
Key Enzyme ◽  
Bivalent Metal Ions ◽  
Ammonium Sulphate Fractionation ◽  
Identification And Characterization

The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner–Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate dehydratase is an octameric protein. The enzyme showed maximal activity at temperatures between 80 and 90 °C and pH values between 6.5 and 7.5, and a half-life of 40 min at 100 °C. Bivalent metal ions such as Co2+, Mg2+, Mn2+ and Ni2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with Km=0.45 mM and Vmax=0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation–dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner–Doudoroff pathway.

scholarly journals Characterization of the binding of plasminogen activators to plasma membranes from human liver

1992 ◽  
Vol 287 (3) ◽  
pp. 911-915 ◽  
Author(s):  
G Nguyen ◽  
S J Self ◽  
C Camani ◽  
E K O Kruithof
Keyword(s):  
Human Liver ◽  
Plasma Membranes ◽  
Gel Filtration ◽  
Mannose Receptor ◽  
Hepatoma Cells ◽  
Tissue Type ◽  
High Affinity ◽  
Liver Membranes ◽  
Membrane Bound ◽  
Pai 1

The binding of tissue-type plasminogen activator (t-PA) to membranes prepared from human liver was investigated, and a specific, saturable, high-affinity binding site (Kd = 3.4 nM) was identified. The binding of t-PA to liver membranes was not affected by an excess of D-mannose or D-galactose, or by active urokinase (u-PA), whereas binding of t-PA to membranes prepared from human HepG2 hepatoma cells was inhibited by u-PA. HepG2-membrane-bound t-PA was fully complexed to PA inhibitor 1 (PAI-1), whereas liver-membrane-bound t-PA was not complexed. Gel filtration on Sephacryl S300 of membrane proteins solubilized in deoxycholate revealed that high-affinity t-PA binding activity elutes at an apparent molecular mass of 40 kDa. Monoclonal antibodies specific for the growth factor and the kringle 2 domains inhibited the binding of t-PA to liver membranes and the catabolism of t-PA by rat hepatoma cells. Human liver membranes also bound u-PA; binding was inhibited by pro-u-PA, the N-terminal fragment of u-PA, but not by the 33 kDa form of u-PA or by t-PA. Our results show that human liver membranes contain a specific 40 kDa binding protein for t-PA that is different from the PAI-1-dependent receptor described on HepG2 cells and the mannose receptor isolated from human liver.

scholarly journals Occurrence of fatty acid epoxide hydrolases in soybean (Glycine max). Purification and characterization of the soluble form

1992 ◽  
Vol 282 (3) ◽  
pp. 711-714 ◽  
Author(s):  
E Blée ◽  
F Schuber
Keyword(s):  
Glycine Max ◽  
Gel Filtration ◽  
Soluble Form ◽  
Soluble Enzyme ◽  
Purification And Characterization ◽  
Epoxide Hydrolases ◽  
Major Activity ◽  
Apparent Homogeneity ◽  
A Minor

Epoxide hydrolases catalysing the hydration of cis-9,10-epoxystearate into threo-9,10-dihydroxystearate have been detected in soybean (Glycine max) seedlings. The major activity was found in the cytosol, a minor fraction being strongly associated with microsomes. The soluble enzyme, which was purified to apparent homogeneity by (NH4)2SO4 fractionation, hydrophobic, DEAE- and gel-filtration chromatographies, has a molecular mass of 64 kDa and a pI of 5.4.

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