scholarly journals Bovine testis and human erythrocytes contain different subtypes of membrane-associated Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphomonoesterases

1994 ◽  
Vol 297 (3) ◽  
pp. 637-645 ◽  
Author(s):  
M Hodgkin ◽  
A Craxton ◽  
J B Parry ◽  
P J Hughes ◽  
B V Potter ◽  
...  
Keyword(s):  
Human Erythrocytes ◽  
Affinity Column ◽  
Type I ◽  
Lower Affinity ◽  
Phorbol Dibutyrate ◽  
Sds Page ◽  
Type Ia ◽  
Erythrocyte Enzyme ◽  
Molecular Masses ◽  
Two Peaks

1. We have purified membrane-associated Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatases from bovine testis and human erythrocytes by chromatography on several media, including a novel 2,3-bisphosphoglycerate affinity column. 2. The enzymes have apparent molecular masses of 42 kDa (testis) and 70 kDa (erythrocyte), as determined by SDS/PAGE, and affinities for Ins(1,4,5)P3 of 14 microM and 22 microM respectively. 3. The two enzymes hydrolyse both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 and are therefore type I Ins(1,4,5)P3 5-phosphatases [nomenclature of Hansen, Johanson, Williamson and Williamson (1987) J. Biol. Chem. 262, 17319-17326]. 4. On chromatofocusing, the partially purified testicular enzyme migrates as two peaks of activity, with pI values of about 5.8 and 5.5. The erythrocyte enzyme exhibits only the latter peak. 5. The testis 5-phosphatase is labile at 37 degrees C, but its activity can be maintained in the presence of 50 mM phorbol dibutyrate (PdBu). After PdBu treatment, a third form of the enzyme, with pI about 6.2, appears on chromatofocusing, but without change in its Km or Vmax. 6. Consideration of the properties of these enzymes and of the 5-phosphatases from other tissues suggests that type I Ins(1,4,5)P3 5-phosphatases are of two well-defined subtypes. We propose that these be termed type Ia [typified by the testis enzyme: approximately 40 kDa, higher affinity for Ins(1,4,5)P3] and Type Ib [typified by the erythrocyte enzyme: approximately 70 kDa, lower affinity for Ins(1,4,5)P3].

scholarly journals Human platelet glycoprotein IIIb binds to thrombospondin fragments bearing the C-terminal region, and/or the type I repeats (CSVTCG motif), but not to the N-terminal heparin-binding region

1992 ◽  
Vol 284 (1) ◽  
pp. 231-236 ◽  
Author(s):  
B Catimel ◽  
L Leung ◽  
H el Ghissasi ◽  
N Mercier ◽  
J McGregor
Keyword(s):  
Plasmodium Falciparum ◽  
Sequence Similarity ◽  
Terminal Region ◽  
Platelet Glycoprotein ◽  
Affinity Column ◽  
Type I ◽  
Heparin Binding ◽  
Western Blots ◽  
Reducing Conditions ◽  
Sds Page

Major blood membrane platelet glycoprotein IIIb (GPIIIb), also termed GPIV or CD365, has been identified as a receptor for thrombospondin (TSP), collagen and Plasmodium falciparum-infected erythrocytes. The aim of the present study was to identify region(s) of TSP involved in binding of GPIIIb. Proteolytic fragments of TSP (M(r) 140 kDa, 120-18 kDa and 27 kDa on SDS/PAGE under reducing conditions) were purified by f.p.l.c. and identified by N-terminal gas-phase sequencing, e.l.i.s.a. and Western blots using monoclonal antibodies directed against defined domains of TSP. The 140 kDa and 120-18 kDa fragments (C-terminal region), but not the 27 kDa fragment (N-terminal region), were shown to bind to GPIIIb by using e.l.i.s.a. and affinity-chromatography systems. TSP binding to a GPIIIb-affinity column was Ca(2+)-dependent and reduced by 45% in the presence of EDTA. Moreover, TSP was only partially eluted with EDTA from a Ca(2+)-equilibrated GPIIIb column. A fragment of 68 kDa, obtained by further digestion of the 140 kDa fragment, bound to the GPIIIb-Sepharose affinity column. This fragment, or stalk-like region, bears the TSP type I repeats that show sequence similarity to regions on properdin, Plasmodium falciparum proteins and antistasin. Peptides (CSVTCG or SVTCGGGV) representing these repeats bound isolated GPIIIb in a Ca(2+)-independent way, but did not completely inhibit the GPIIIb and TSP interaction. These studies indicate that GPIIIb binds to the TSP via the C-terminal region and/or the CSVTCG motif, but not to the N-terminal region. Interaction between GPIIIb and the TSP C-terminal region or the CSVTCG motif is respectively Ca(2+)-dependent and -independent.

scholarly journals Purification and characterization of a connective-tissue-degrading metalloproteinase from the cytosol of metastatic melanoma cells

1987 ◽  
Vol 245 (2) ◽  
pp. 429-437 ◽  
Author(s):  
S Zucker ◽  
T Turpeenniemi-Hujanen ◽  
N Ramamurthy ◽  
J Wieman ◽  
R Lysik ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Type I Collagen ◽  
Collagen Type I ◽  
Enzymic Activity ◽  
Affinity Column ◽  
Type I ◽  
Type Iv ◽  
Affinity Column Chromatography ◽  
Molecular Masses

A metalloproteinase with activity against type IV collagen, type I collagen and gelatin has been purified from the cytosol of a highly metastatic mouse melanoma by anion-exchange, zinc-chelated and lectin-affinity column chromatography. The purified enzyme has a molecular mass of approx. 59 kDa and on isoelectric focusing in two-dimensional gels produced three spots with apparent isoelectric points (pI) between 5.7 and 6.1. Enzymic activity with collagen, but not gelatin, substrates was latent, requiring activation by trypsin or organomercurials. Trypsin activation of this metalloproteinase was accompanied by a change in molecular mass, whereas autoactivation after 1 month's storage, was not. The degradation of types I and IV collagen by the melanoma enzyme yielded products of lower molecular masses than those yielded by mammalian collagenases, this characteristic thus differentiating this metalloproteinase from classical collagenases.

Changes in the activities of protein phosphatase type 1 and type 2A in sea urchin embryos during early development

Zygote ◽  
1999 ◽  
Vol 8 (S1) ◽  
pp. S68-S69
Author(s):  
Manabu Kawamoto ◽  
Akiko Fujiwara ◽  
Shin-ichi Kuno ◽  
Ikuo Yasumasu
Keyword(s):  
Sea Urchin ◽  
Mammalian Cells ◽  
Cell Movement ◽  
Catalytic Subunit ◽  
Sea Urchin Eggs ◽  
Nitrophenyl Phosphate ◽  
Sds Page ◽  
Molecular Masses ◽  
Two Peaks

Serine/threonine protein phosphatases expected to participate in the process of signal transduction, cell movement such as cell division and gene expression (Kinoshita et al., 1990; Healy et al., 1991; Mayer-Jaekel et al., 1993; Mumby & Walter, 1993), are classified into type 1 (PP1), type 2A (PP2A), type 2B and type 2C in mammalian cells. PP1 and PP2A are known to be strongly inhibited by okadaic acid (OA) (Tachibana et al., 1981; Bialojan Takai, 1988), a polyether fatty acid isolated from the marine sponge Halicondria okadai (Haystead et al., 1989). OA is also known to inhibit PP2A at lower concentrations than that to block PP1 in mammalian cells, but does not inhibit the activities of other phosphatase species (Ishihara et al., 1989).The p-nitrophenyl phosphate (pNPP) splitting activity in the extract obtained from eggs of the sea urchin Hemicentrotus pulcherrimus was found to be inhibited by OA and calyculin A (CLA), potent inhibitors of PP1 and PP2A. OA-sensitive phosphatases are known to catalyse pNPP splitting (Takai & Mieskes, 1991), in the same manner as other OA-insensitive phosphatases.Four peaks of the pNPP splitting activity were obtained by QAE-Toyopearl chromatography in the extract of sea urchin eggs. In two of these four peaks, pNPP splitting reactions were strongly inhibited by OA and CLA at quite low concentration. High sensitivities of the pNPP splitting reaction to OA and CLA in these two peaks suggest that pNPP splitting results from the reaction catalysed by PP2A. The molecular masses of proteins exhibiting OA-sensitive pNPP splitting activities in these two peaks were found to be about 160 kDa by Superdex 200HR, and were similar to that of mammalian PP2A trimeric holoenzyme. By immunoblot analyses with anti-human PP2A catalytic subunit antibody, an immunoreactive 36 kDa protein was found by SDS-PAGE in a peak of OA-sensitive pNPP splitting activity obtained by QAE-Toyopearl chromatography. Sea urchin eggs have at least two PP2A-like enzymes with similar molecular masses to that of mammalian PP2A, and one of them contains human-type catalytic subunit.

Purification and characterization of activated human erythrocyte prolidase

1989 ◽  
Vol 67 (1) ◽  
pp. 34-41 ◽  
Author(s):  
Andrea M. Richter ◽  
Gerald L. Lancaster ◽  
Francis Y. M. Choy ◽  
Peter Hechtman
Keyword(s):  
Molecular Weight ◽  
Liquid Chromatography ◽  
Human Kidney ◽  
Human Erythrocytes ◽  
Rabbit Antibody ◽  
Purification And Characterization ◽  
Sds Page ◽  
Erythrocyte Enzyme ◽  
Chromatography Columns

Prolidase (E.C. 3.4.13.9) has been purified 7500-fold to homogeneity from human erythrocytes in a Mn2+-activated form using conventional and fast protein liquid chromatography columns. The procedure includes a 1-h incubation of the crude hemolysate at 50 °C with 1 mM MnCl2. Following this novel step, prolidase retains full activity, obviating the requirement for preincubation of each enzyme fraction with Mn2+ prior to assay. Preincubation with MnCl2 does not change the isoelectric point of the enzyme. The molecular weight of the purified enzyme was 58 000 when measured by SDS–PAGE. Western blotting, using rabbit antibody raised to human kidney prolidase, with partially purified erythrocyte enzyme revealed a cross-reacting band at Mr 58 000.Key words: prolidase purification, human erythrocytes.

scholarly journals CAMP factor homologues in Propionibacterium acnes: a new protein family differentially expressed by types I and II

Microbiology ◽  
2005 ◽  
Vol 151 (5) ◽  
pp. 1369-1379 ◽  
Author(s):  
Susanna Valanne ◽  
Andrew McDowell ◽  
Gordon Ramage ◽  
Michael M. Tunney ◽  
Gisli G. Einarsson ◽  
...  
Keyword(s):  
Silver Staining ◽  
Type Strain ◽  
Propionibacterium Acnes ◽  
Draft Genome ◽  
Pcr Amplification ◽  
Opportunistic Pathogen ◽  
Camp Factor ◽  
Sds Page ◽  
Type Ia ◽  
Molecular Masses

Analysis of the draft genome sequence of the opportunistic pathogen Propionibacterium acnes type strain NCTC 737 (=ATCC 6919) revealed five genes with sequence identity to the co-haemolytic Christie–Atkins–Munch-Peterson (CAMP) factor of Streptococcus agalactiae. The predicted molecular masses for the expressed proteins ranged from 28 to 30 kDa. The genes were present in each of the three recently identified recA-based phylogenetic groupings of P. acnes (IA, IB and II), as assessed by PCR amplification. Conserved differences in CAMP factor gene sequences between these three groups were also consistent with their previous phylogenetic designations. All type IA, IB and II isolates were positive for the co-haemolytic reaction on sheep blood agar. Immunoblotting and silver staining of SDS-PAGE gels, however, revealed differential protein expression of CAMP factors amongst the different groups. Type IB and II isolates produced an abundance of CAMP factor 1, detectable by specific antibody labelling and silver staining of SDS-PAGE gels. In contrast, abundant CAMP factor production was lacking in type IA isolates, although larger amounts of CAMP factor 2 were detectable by immunoblotting compared with type II isolates. While the potential role of the abundant CAMP factor 1 in host colonization or virulence remains to be determined, it should be noted that the type strain of P. acnes used in much of the published literature is a type IA isolate and is, therefore, lacking in this attribute.

Detection of Carriers in Glanzmann's Thrombasthenia

1983 ◽  
Vol 49 (03) ◽  
pp. 182-186
Author(s):  
G T E Zonneveld ◽  
E F van Leeuwen ◽  
A Sturk ◽  
J W ten Cate
Keyword(s):  
Bleeding Time ◽  
Periodic Acid ◽  
Type I ◽  
Clot Retraction ◽  
Periodic Acid Schiff ◽  
Glanzmann’S Thrombasthenia ◽  
Aggregation Response ◽  
Glanzmann's Thrombasthenia ◽  
Sds Page ◽  
Reduced State

SummaryQuantitative glycoprotein (GP) analysis of whole platelets or platelet membranes was performed by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) and periodic acid Schiff staining in the families of two unrelated Glanzmann’s thrombasthenia (GT) patients. Each family consisted of two symptom free parents, a symptom free daughter and a GT daughter. All symptom free members had a normal bleeding time, clot retraction and platelet aggregation response to adenosine 5’-diphosphate (ADP), collagen and adrenalin. Platelet Zw* antigen was normally expressed in these subjects. GT patiens, classified as a type I and II subject, showed reduced amounts of GP lib and of GP nia. Analysis of isolated membranes in the non-reduced state, however, showed that the amount of GP Ilia was also reduced in three of the four parents, whereas one parent (of the GT type I patient) and the two unaffected daughters had normal amounts of GP Ilia. Quantitative SDS-PAGE may therefore provide a method for the detection of asymptomatic carriers in GT type I and II.

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.

scholarly journals Relative rates of biosynthesis of collagen type I, type V and type VI in calf cornea

1991 ◽  
Vol 274 (2) ◽  
pp. 615-617 ◽  
Author(s):  
P Kern ◽  
M Menasche ◽  
L Robert
Keyword(s):  
Type I Collagen ◽  
Collagen Type ◽  
Corneal Stroma ◽  
Collagen Type I ◽  
Type I ◽  
Type Vi Collagen ◽  
Sds Page ◽  
Proline Incorporation ◽  
Type V

The biosynthesis of type I, type V and type VI collagens was studied by incubation of calf corneas in vitro with [3H]proline as a marker. Pepsin-solubilized collagen types were isolated by salt fractionation and quantified by SDS/PAGE. Expressed as proportions of the total hydroxyproline solubilized, corneal stroma comprised 75% type I, 8% type V and 17% type VI collagen. The rates of [3H]proline incorporation, linear up to 24 h for each collagen type, were highest for type VI collagen and lowest for type I collagen. From pulse-chase experiments, the calculated apparent half-lives for types I, V and VI collagens were 36 h, 10 h and 6 h respectively.

scholarly journals The study of multipeaked type-I X-ray bursts in the neutron star low-mass X-ray binary 4U 1636 − 536 with RXTE

2020 ◽  
Vol 501 (1) ◽  
pp. 168-178
Author(s):  
Chen Li ◽  
Guobao Zhang ◽  
Mariano Méndez ◽  
Jiancheng Wang ◽  
Ming Lyu
Keyword(s):  
Neutron Star ◽  
Flux Ratio ◽  
Light Curves ◽  
Type I ◽  
X Ray ◽  
Soft State ◽  
Peak Flux ◽  
Low Mass ◽  
Two Peaks ◽  
Second Peak

ABSTRACT We have found and analysed 16 multipeaked type-I bursts from the neutron-star low-mass X-ray binary 4U 1636 − 53 with the Rossi X-ray Timing Explorer (RXTE). One of the bursts is a rare quadruple-peaked burst that was not previously reported. All 16 bursts show a multipeaked structure not only in the X-ray light curves but also in the bolometric light curves. Most of the multipeaked bursts appear in observations during the transition from the hard to the soft state in the colour–colour diagram. We find an anticorrelation between the second peak flux and the separation time between two peaks. We also find that in the double-peaked bursts the peak-flux ratio and the temperature of the thermal component in the pre-burst spectra are correlated. This indicates that the double-peaked structure in the light curve of the bursts may be affected by enhanced accretion rate in the disc, or increased temperature of the neutron star.

Application of 2D BN/SDS-PAGE coupled with mass spectrometry for identification of VDAC-associated protein complexes related to mitochondrial binding sites for type I brain hexokinase

Mitochondrion ◽  
2013 ◽  
Vol 13 (6) ◽  
pp. 823-830 ◽  
Author(s):  
Carla Rossini Crepaldi ◽  
Phelipe Augusto Mariano Vitale ◽  
Andrea Cristina Tesch ◽  
Hélen Julie Laure ◽  
José César Rosa ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Binding Sites ◽  
Protein Complexes ◽  
Type I ◽  
Sds Page
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