Identification and Genetic Analysis of a Common Molecular Variant of Histidine-rich Glycoprotein with a Difference of 2KD in Apparent Molecular Weight

1995 ◽  
Vol 74 (06) ◽  
pp. 1491-1496 ◽  
Author(s):  
B C Hennis ◽  
P A van Boheemen ◽  
S Wakabayashi ◽  
T Koide ◽  
J J M L Hoffmann ◽  
...  
Keyword(s):  
Consensus Sequence ◽  
Apparent Molecular Weight ◽  
Mendelian Inheritance ◽  
Glycosylation Site ◽  
Single Chain ◽  
Chain Molecules ◽  
Molecular Weights ◽  
Molecular Variants ◽  
Direct Sequence Analysis ◽  
The Difference

SummaryTwo forms of histidine-rich glycoprotein (HRG) were detected on SDS-PAGE by silver staining and immunoblotting after isolation of the protein from pooled plasma using immuno-affinity chromatography followed by chromatography with heparin-Sepharose. Both forms were single-chain molecules and the apparent molecular weights of form 1 and form 2 were 77 kD and 75 kD respectively. Mendelian inheritance of both HRG forms was observed in four families with 24 informative meioses, strongly suggesting that the two forms are encoded by different alleles. The frequency of form 1 and form 2 in a group of 36 individuals was 0.35 and 0.65 respectively.The difference between the two molecular variants was studied by direct sequence analysis of amplified exons of the HRG gene from 6 individuals who were homozygous either for form 1 or form 2. Five amino acid polymorphisms in three different exons were observed: Ile/Thr in exon 4; Pro/Ser in exon 5; His/Arg, Arg/Cys and Asn/Ile in exon 7. Analysis of these polymorphisms in 20 volunteers showed that only the Pro/Ser polymorphism at position 186 in exon 5 was coupled to the form of the HRG protein. Ser was found in form 1 and Pro in form 2. The presence of Ser at position 186 introduces a consensus sequence for a N-glycosylation site (Asn-X-Ser/Thr). By removing N-linked sugars with N-glycanase, it could be demonstrated that the difference between the two forms of HRG is caused by an extra carbohydrate group at Asn 184 in form 1.

Purification and properties of two phospholipid transfer proteins from yeast

1987 ◽  
Vol 65 (3) ◽  
pp. 195-202 ◽  
Author(s):  
Richard P. Bozzato ◽  
David O. Tinker
Keyword(s):  
High Performance ◽  
Apparent Molecular Weight ◽  
Cellular Growth ◽  
Yeast Saccharomyces Cerevisiae ◽  
Anion Exchange Column ◽  
Molecular Weights ◽  
Transfer Protein ◽  
Isolation And Characterization ◽  
Phospholipid Transfer ◽  
The Difference

Several intracellular proteins of low and intermediate molecular weights have been isolated from a variety of mammalian and plant tissues that possess an ability to catalyze the transfer or exchange of intact phospholipid molecules between different membrane systems. The soluble cytosolic fraction of the yeast Saccharomyces cerevisiae also contains phospholipid transfer activity that varies with both the state of cellular growth and the type of metabolic carbon source. This activity is protein in nature and very unstable, and requires powerful separation techniques for its purification. Here we report the isolation and characterization of two phospholipid transfer proteins from yeast, one of which we believe represents a partial proteolytic product of the other. The two proteins were purified to near homogeneity through a combination of dye–ligand and high performance ion-exchange chromatographic techniques. Transfer protein I (TP-I) is eluted at a lower ionic strength from an anion-exchange column than transfer protein II (TP-II), which reflects the difference in their isoelectric points; TP-I has a pI of 6.3, while that for TP-II is 6.1. Both species have the same apparent molecular weight of 33 400 and virtually identical substrate specificities. The order of the relative rates of phospholipid transfer are phosphatidylcholine > phosphatidylethanolamine > phosphatidylinositol > phosphatidylserine.

Inhibition in Purified Systems and in Human Plasma of Chimaeric Plasminogen Activators Consisting of the NH2-Terminal Region of Tissue-Type Plasminogen Activator and the COOH-Terminal Region of UrokinaseType Plasminogen Activator

1988 ◽  
Vol 60 (02) ◽  
pp. 247-250 ◽  
Author(s):  
H R Lijnen ◽  
L Nelles ◽  
B Van Hoef ◽  
F De Cock ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Rate Constants ◽  
Plasminogen Activators ◽  
Second Order ◽  
Tissue Type ◽  
Single Chain ◽  
Chain Molecules ◽  
Order Rate ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

SummaryRecombinant chimaeric molecules between tissue-type plasminogen activator (t-PA) and single chain urokinase-type plasminogen activator (scu-PA) or two chain urokinase-type plasminogen activator (tcu-PA) have intact enzymatic properties of scu-PA or tcu-PA towards natural and synthetic substrates (Nelles et al., J Biol Chem 1987; 262: 10855-10862). In the present study, we have compared the reactivity with inhibitors of both the single chain and two chain variants of recombinant u-PA and two recombinant chimaeric molecules between t-PA and scu-PA (t-PA/u-PA-s: amino acids 1-263 of t-PA and 144-411 of u-PA; t-PA/u-PA-e: amino acids 1-274 of t-PA and 138-411 of u-PA). Incubation with human plasma in the absence of a fibrin clot for 3 h at 37° C at equipotent concentrations (50% clot lysis in 2 h), resulted in significant fibrinogen breakdown (to about 40% of the normal value) for all two chain molecules, but not for their single chain counterparts. Preincubation of the plasminogen activators with plasma for 3 h at 37° C, resulted in complete inhibition of the fibrinolytic potency of the two chain molecules but did not alter the potency of the single chain molecules. Inhibition of the two chain molecules occurred with a t½ of approximately 45 min. The two chain variants were inhibited by the synthetic urokinase inhibitor Glu-Gly-Arg-CH2CCl with apparent second-order rate constants of 8,000-10,000 M−1s−1, by purified α2-antiplasmin with second-order rate constants of about 300 M−1s−1, and by plasminogen activator inhibitor-1 (PAI-1) with second-order rate constants of approximately 2 × 107 M−1s−1.It is concluded that the reactivity of single chain and two chain forms of t-PA/u-PA chimaers with inhibitors is very similar to that of the single and two chain forms of intact u-PA.

Detection of both Type 1 and Type 2 Plasminogen Activator Inhibitors in Human Monocytes

1990 ◽  
Vol 63 (01) ◽  
pp. 067-071 ◽  
Author(s):  
Joan C Castellote ◽  
Enric Grau ◽  
Maria A Linde ◽  
Nuria Pujol-Moix ◽  
Miquel LI Rutllant
Keyword(s):  
Molecular Mass ◽  
Plasminogen Activator ◽  
Human Peripheral Blood ◽  
Direct Interaction ◽  
Apparent Molecular Weight ◽  
Fibrinolytic System ◽  
Human Monocytes ◽  
Blood Monocytes ◽  
Single Chain ◽  
Sds Page

SummaryIncreasing evidence suggests the involvement of leukocytes in the fibrinolytic system. Monocytes secrete pro-urokinase (Grau, Thromb Res 1989; 53: 145) and it has been shown that these cells have specific receptors for urokinase and plasminogen (Miles, Thromb Haemostas 1987; 58: 936). The aim of this study was to analyse the presence of plasminogen activator inhibitor(s) in platelet-free suspensions of human peripheral blood monocytes and polymorphonuclear leukocytes (PMN). SDS-PAGE and reverse fibrin autography showed an inhibitory band of 50 kDa in the monocyte extracts (Triton X-100) but not in the PMN extracts. Urokinase (u-PA) was mixed with increasing amounts of monocyte extract for 10 min and the mixtures were added to 125Ifibrin coated wells containing plasminogen. A dose-dependent decrease in the u-PA fibrinolytic activity was observed. The amount of inhibition increased when the monocyte releasates were preincubated with u-PA (40% inhibition after 5 min preincubation and 80% after 15 min), indicating a direct interaction between this activator and an inhibitor(s). After SDS-PAGE of monocyte extracts, immunoblotting and peroxidase staining identified both PAI1 and PAI2, with an apparent molecular weight of 47-50 kDa. Monocyte-associated PAI1 formed complexes with single chain t-PA with a molecular mass 50 kDa higher than the molecular mass of the free PAI1. However, a significant amount of PAI remained unbound to t-PA. This inactive PAI1 could have come from a rapid inactivation of the primary active PAI1. These PAI1 and PAI2 detected in human monocytes may be transcendent in the regulation of the fibrinolytic system.

Molecular Forms of Human Protein C: Comparison and Distribution in Human Adult Plasma

1989 ◽  
Vol 62 (03) ◽  
pp. 902-905 ◽  
Author(s):  
Brian S Greffe ◽  
Marilyn J Manco-Johnson ◽  
Richard A Marlar
Keyword(s):  
Heavy Chain ◽  
Protein C ◽  
Molecular Forms ◽  
Post Translational Modification ◽  
Single Chain ◽  
Beta Form ◽  
Sds Page ◽  
Dependent Protein ◽  
Adult Plasma ◽  
The Difference

SummaryProtein C (PC) is a vitamin K-dependent protein which functions as both an anticoagulant and profibrinolytic. It is synthesized as a single chain protein (SC-PC) and post-transla-tionally modified into a two chain form (2C-PC). Two chain PC consists of a light chain (LC) and a heavy chain (HC). The present study was undertaken to determine the composition of the molecular forms of PC in plasma. PC was immunoprecipitated, subjected to SDS-PAGE and Western blotting. The blots were scanned by densitometry to determine the distribution of the various forms. The percentage of SC-PC and 2C-PC was found to be 10% and 90% respectively. This is in agreement with previous work. SC-PC and the heavy chain of 2C-PC consisted of three molecular forms (“alpha”, “beta”, and “gamma”). The “alpha” form of HC is the standard 2C form with a MW of 40 Kd. The “beta” form of HC has also been described and has MW which is 4 Kd less than the “alpha” form. The “gamma” species of the SC and 2C-PC has not been previously described. However, its 3 Kd difference from the “beta” form could be due to modification of the “beta” species or to a separate modification of the alpha-HC. The LC of PC was shown to exist in two forms (termed form 1 and form 2). The difference between these two forms is unknown. The molecular forms of PC are most likely due to a post-translational modification (either loss of a carbohydrate or a peptide) rather than from plasma derived degradation.

scholarly journals A proteoglycan form of heparin and its degradation to single-chain molecules.

1978 ◽  
Vol 253 (19) ◽  
pp. 6687-6693 ◽  
Author(s):  
H.C. Robinson ◽  
A.A. Horner ◽  
M. Höök ◽  
S. Ogren ◽  
U. Lindahl
Keyword(s):  
Single Chain ◽  
Chain Molecules

scholarly journals Nuclear matrix of HeLa S3 cells. Polypeptide composition during adenovirus infection and in phases of the cell cycle.

1977 ◽  
Vol 72 (1) ◽  
pp. 194-208 ◽  
Author(s):  
L D Hodge ◽  
P Mancini ◽  
F M Davis ◽  
P Heywood
Keyword(s):  
Cell Cycle ◽  
Molecular Weight ◽  
Nuclear Matrix ◽  
Apparent Molecular Weight ◽  
Molecular Weight Range ◽  
Weight Range ◽  
Molecular Weights ◽  
Liver Nuclei ◽  
Hela S3 ◽  
Biochemical Analyses

A subnuclear fraction has been isolated from HeLa S3 nuclei after treatment with high salt buffer, deoxyribonuclease, and dithiothreitol. This fraction retains the approximate size and shape of nuclei and resembles the nuclear matrix recently isolated from rat liver nuclei. Ultrastructural and biochemical analyses indicate that this structure consists of nonmembranous elements as well as some membranous elements. Its chemical composition is 87% protein, 12% phospholipid, 1% DNA, and 0.1% RNA by weight. The protein constituents are resolved in SDS-polyacrylamide slab gels into 30-35 distinguishable bands in the apparent molecular weight range of 14,000 - 200,000 with major peptides at 14,000 - 18,000 and 45,000 - 75,000. Analysis of newly synthesized polypeptides by cylindrical gel electrophoresis reveals another cluster in the 90,000-130,000 molecular weight range. Infection with adenovirus results in an altered polypeptide profile. Additional polypeptides with apparent molecular weights of 21,000, 23,000, and 92,000 become major components by 22 h after infection. Concomitantly, some peptides in the 45,000-75,000 mol wt range become less prominent. In synchronized cells the relative staining capacity of the six bands in the 45,000-75,000 mol wt range changes during the cell cycle. Synthesis of at least some matrix polypeptides occures in all phases of the cell cycle, although there is decreased synthesis in late S/G2. In the absence of protein synthesis after cell division, at least some polypeptides in the 45,000-75,000 mol wt range survive nuclear dispersal and subsequent reformation during mitosis. The possible significance of this subnuclear structure with regard to structure-function relationships within the nucleus during virus replication and during the life cycle of the cell is discussed.

scholarly journals Structural Analysis of Yoked Chorionic Gonadotropin-Luteinizing Hormone Receptor Ectodomain Complexes by Circular Dichroic Spectroscopy

2003 ◽  
Vol 17 (7) ◽  
pp. 1192-1202 ◽  
Author(s):  
Gregory B. Fralish ◽  
Brian Dattilo ◽  
David Puett
Keyword(s):  
Chorionic Gonadotropin ◽  
Fusion Proteins ◽  
Homology Model ◽  
Difference Spectrum ◽  
Luteinizing Hormone Receptor ◽  
Amino Acid Residues ◽  
Single Chain ◽  
Glycoprotein Hormone ◽  
The Difference ◽  
Α Helix

Abstract Binding of the heterodimeric glycoprotein hormone, chorionic gonadotropin (CG), occurs to the heptahelical LH receptor N-terminal ectodomain (ECD), a large portion of which has been modeled as a leucine-rich repeat protein. In this study, we expressed and purified three single chain N-CG-ECD-C complexes, one comprising the full-length ECD, 1–341 (encoded by exons 1–10 and a portion of 11), and two C-terminal ECD deletion fragments, 1–294 (encoded by exons 1–10) and 1–180 (encoded by exons 1–7). The fusion proteins, including yoked CG (N-β-α-C), were characterized by Western blot analysis and circular dichroism (CD). Analysis of the CD spectra obtained on the CG-ECD fusion proteins, and of the difference spectrum of each after subtracting the CG contribution, yielded secondary structures consistent with a repeating β-strand/α-helix fold as predicted in the homology model. A marked decrease in helicity was observed when the C-terminal 47 amino acid residues were removed from the ECD. Removal of an additional 114 residues, i.e. the region encoded by exons 8–10, results in the loss of fewer helical residues. These results suggest that the hinge region of the ECD, predicted to contain only limited secondary structure, interacts with and stabilizes the ligand-occupied N-terminal portion. Furthermore, the results support a repeating fold, consistent with the proposed model for the LHR ECD.

A PLASMINOGEN ACTIVATOR INHIBITOR (PAI-2) CIRCULATES IN TWO HIGH MOLECULAR WEIGHT FORMS IN PREGNANCY

1987 ◽  
Author(s):  
N A Booth ◽  
A Reith ◽  
B Bennett
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Plasminogen Activator ◽  
Apparent Molecular Weight ◽  
Late Pregnancy ◽  
Plasminogen Activator Inhibitor ◽  
Molecular Weights ◽  
Histiocytic Cell ◽  
Sds Page ◽  
Pai 1

Normal vascular endothelium and platelet α-granules contain an inhibitor of plasminogen activator (PAI-1) of about 48000 molecular weight, which is released by stimuli such as thrombin. An immunologically distinct inhibitor (PAI-2) of about 47000 molecular weight has been purified from placenta and from a histiocytic cell line U-937. The level of PA-inhibition in plasma is raised in late pregnancy and this may be due to increases in PAI-1 or in PAI-2 or in both.Using SDS-PAGE and zymography on fibrin/plasminogen /u-PA detector gels, we have found that normal plasma contains a band of inhibition of apparent molecular weight 40000, which can be neutralised by antiserum raised against PAI-1. Pregnancy plasma contained this band as well as additional inhibitor bands of apparent molecular weights 75000 and 130000. The novel high molecular weight PA-inhibitors were detectable by zymography at about 12 weeks gestation. They were specific for plasminogen activator and did not inhibit plasmin. They were inhibited by antiserum raised against PAI-2 from U-937 cells (a gift from Dr EKO Kruithof) and thus are immunologically related to PAI-2. They may represent circulating complexes of PAI-2 with another protein or aggregates of PAI-2, which retain inhibitory activity after SDS-PAGE. PAI-2 appears to represent a pregnancy associated protein that circulates in a number of different molecular weight forms.

scholarly journals On the Difference between Self-Assembling Process of Monomeric and Dimeric Surfactants with the Same Head to Tail Ratio: A Lattice Monte Carlo Simulation

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Reza Behjatmanesh-Ardakani ◽  
Maryam Farsad
Keyword(s):  
Monte Carlo Simulation ◽  
Monte Carlo ◽  
Gemini Surfactants ◽  
Chain Structure ◽  
Single Chain ◽  
Lattice Monte Carlo ◽  
Self Assembling ◽  
Spacer Group ◽  
The Difference ◽  
Dimeric Surfactants

Experimental data show that gemini surfactants have critical micelle concentrations that are almost tenfold lower than the CMCs of single chain ones. It is believed that the spacer groups play an important role in this subject. Short hydrophilic or long hydrophobic spacers can reduce CMC dramatically. In this paper, self-assembling processes of double-chain and one-chain surfactants with the same head to tail ratio are compared. Dimeric chain structure is exactly double of single chain. In other words, hydrophilic-lyophilic balances of two chain models are the same. Two single chains are connected head-to-head to form a dimeric chain, without introducing extra head or tail beads as a spacer group. Premicellar, micellar, and shape/phase transition ranges of both models are investigated. To do this, lattice Monte Carlo simulation in canonical ensemble has been used. Results show that without introducing extra beads as spacer group, the CMC of (H3T3)2as a dimeric surfactant is much lower than the CMC of its similar single chain, H3T3. For dimeric case of study, it is shown that bolaform aggregates are formed.

scholarly journals Isolation of palmitoyl-CoA hydrolases from human blood platelets

1981 ◽  
Vol 199 (3) ◽  
pp. 639-647 ◽  
Author(s):  
R K Berge ◽  
L E Hagen ◽  
M Farstad
Keyword(s):  
Human Blood ◽  
Blood Platelets ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Cytosol Fraction ◽  
Molecular Weights ◽  
Apparent Homogeneity ◽  
Coa Hydrolase ◽  
Human Blood Platelets

The palmitoyl-CoA hydrolase activity, which in human blood platelets is mainly localized in the cytosol fraction [Berge, Vollset & Farstad (1980) Scand. J. Clin. Lab. Invest. 40, 271--279], was found to be extremely labile. Inclusion of glycerol or palmitoyl-CoA stabilized the activity during preparation. Gel-filtration studies revealed multiple forms of the enzyme with molecular weights corresponding to about 70 000, 40 000 and 24 000. The relative recovery of the mol.wt.-70 000 form was increased by the presence of 20% (v/v) glycerol or 10 microM-palmitoyl-CoA. The three enzyme forms are probably unrelated, since they were not interconvertible. The three different species of palmitoyl-CoA hydrolase were purified by DEAE-cellulose and hydroxyapatite chromatography, isoelectric focusing and high-pressure liquid chromatography (h.p.l.c.) to apparent homogeneity. The three enzymes had isoelectric points (pI) of 7.0, 6.1 and 4.9. The corresponding molecular weights were 27 000--33 000, 66 000--72 000 and 45 000--49 000, calculated from h.p.l.c. and Ultrogel AcA-44 chromatography. The apparently purified enzymes were unstable, as most of the activity was lost during purification. The enzyme with an apparent molecular weight of 45 000--49 000 was split into fractions with molecular weights of less than 10 000 by re-chromatography on h.p.l.c. concomitantly with a loss of activity. The stimulation of the activity by the presence of serum albumin seems to depend on the availability of palmitoyl-CoA, as has been reported for other palmitoyl-CoA hydrolases. [Berge & Farstad (1979) Eur. J. Biochem. 96, 393--401].

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