Sialic Acid Dependent Polypeptide Chain Heterogeneity of Human Fibrinogen Demonstrated by Two-Dimensional Electrophoresis

SummaryHuman fibrinogen was compared with asialofibrinogen by two-dimensional electrophoresis to evaluate the contribution of sialic acid to the heterogeneity of the γ- and Bβ-polypeptide chains.Reduced fibrinogen showed three major variants for both the γ- and Bβ-chains. In addition two minor γ-bands with a more acidic isoelectric point than the normal γ-chains were observed. Electrophoresis in the second dimension (SDS) suggests that these most acidic bands are γ-chain-variants with a higher molecular weight. In asialofibrinogen only two predominant variants with more alkaline isoelectric points were present in each chain type.It is concluded that enzymatic removal of sialic acid partially reduces the heterogeneity of the γ- and Bβ-polypeptide chains of human fibrinogen, but additional sources producing charge heterogeneity must be sought.

Download Full-text

Analytical electrofocusing and two-dimensional electrophoresis of proteins extracted from the mycelia of aggressive and nonaggressive strains of Ophiostoma ulmi

Canadian Journal of Botany ◽

10.1139/b86-272 ◽

1986 ◽

Vol 64 (9) ◽

pp. 2073-2081 ◽

Cited By ~ 10

Author(s):

Robert S. Jeng

Keyword(s):

Dutch Elm Disease ◽

Two Dimensional ◽

Polyacrylamide Gels ◽

Ophiostoma Ulmi ◽

Two Dimensional Electrophoresis ◽

Coomassie Blue ◽

Isoelectric Points ◽

Additional Protein ◽

Dimensional Electrophoresis ◽

Electrophoresis Of Proteins

Soluble mycelial proteins from Ophiostoma ulmi (Buism.) Nannf., the causal agent of Dutch elm disease, were separated by analytical electrofocusing and two-dimensional electrophoresis in polyacrylamide gels. Results showed the aggressive and nonaggressive strains of this pathogen each had about 60 Coomassie blue stained bands having isoelectric points from 3 to 7. Both strains of this fungus had their own characteristic electrofocusing patterns. Nonaggressive isolate S116, for example, lacked two protein bands, one near the anode and one near the cathode, but it had five additional protein bands distributed from pH 4 to 6. Two-dimensional electrophoresis of total soluble proteins depicted that there were 36 proteins found to be specific for the nonaggressive isolate S116 and 12 proteins for the aggressive isolate RR2.

Download Full-text

Plasma apolipoproteins in Tangier disease, as studied with two-dimensional electrophoresis.

Clinical Chemistry ◽

10.1093/clinchem/33.1.120 ◽

1987 ◽

Vol 33 (1) ◽

pp. 120-122 ◽

Cited By ~ 8

Author(s):

S Visvikis ◽

M F Dumon ◽

J Steinmetz ◽

T Manabe ◽

M M Galteau ◽

...

Keyword(s):

The Other ◽

High Density Lipoproteins ◽

Major Protein ◽

Two Dimensional ◽

Tangier Disease ◽

Two Dimensional Electrophoresis ◽

Isoelectric Points ◽

Molecular Masses ◽

Dimensional Electrophoresis ◽

Homozygous Patient

Abstract Tangier disease is characterized by a deficiency of high-density lipoproteins and of their major protein constituent, apolipoprotein (apo) A-I. We used high-resolution two-dimensional electrophoresis to examine the principal plasma apolipoproteins (A-I, A-II, A-IV, E, C-II, and C-III) of three persons with Tangier disease, one homozygous patient and his two heterozygous children, comparing the patterns with those for healthy subjects. Characteristic abnormalities were found in the distribution of the isoproteins of apo A-I, there being a normal concentration of pro apo A-I but dramatically decreased concentrations of the other apo A-I isoproteins. We also found hitherto-undescribed polypeptide abnormalities in apo C-III: sialylated and nonsialylated forms of apo C-III appear as double spots having the same isoelectric points but different molecular masses. No other substantial difference was detected in the polypeptide distribution of the other plasma apolipoproteins.

Download Full-text

High Resolution Electrophoretic Analysis of Human Fibrinogen and Its Crosslinked Intermediates

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657313 ◽

1983 ◽

Vol 49 (01) ◽

pp. 047-050 ◽

Cited By ~ 5

Author(s):

N A Carrell ◽

J R Holahan ◽

G C White ◽

J McDonagh

Keyword(s):

High Resolution ◽

Electrophoretic Analysis ◽

Two Dimensional ◽

Charge Heterogeneity ◽

Human Fibrinogen ◽

Striking Increase ◽

Single Donor ◽

Isoelectric Points ◽

Fraction I ◽

Electrophoretic Procedure

SummaryHeterogeneity in human fibrinogen was examined using an improved two-dimensional isoelectric focusing-SDS polyacrylamide gel electrophoretic procedure. Four different preparations of fibrinogen were compared: single donor fibrinogen prepared from plasma by precipitation with ammonium sulfate or by affinity chromatography on fibrin-monomer Sepharose, fraction I—4 prepared from Cohn fraction I paste, and Kabi grade L. The subunit Aα, Bβ, and γ chains in all preparations had marked charge heterogeneity. The three chains were clearly separated from each other and a range of isoelectric points for each chain could be assigned. Minor variations in the subunit heterogeneity of the different preparations were found. Intermediates in the transition from fibrinogen to crosslinked fibrin were also examined. A striking increase in the heterogeneity of the α chain was observed during crosslinking.

Download Full-text

Muscle protein analysis. I. High-resolution two-dimensional electrophoresis of skeletal muscle proteins for analysis of small biopsy samples.

Clinical Chemistry ◽

10.1093/clinchem/25.11.1877 ◽

1979 ◽

Vol 25 (11) ◽

pp. 1877-1884 ◽

Cited By ~ 57

Author(s):

C S Giometti ◽

N G Anderson ◽

N L Anderson

Keyword(s):

High Resolution ◽

Muscle Protein ◽

Large Fraction ◽

Psoas Muscle ◽

Human Muscle ◽

Rabbit Muscle ◽

Two Dimensional ◽

Charge Heterogeneity ◽

Two Dimensional Electrophoresis ◽

Dimensional Electrophoresis

Abstract We have been developing a clinically useful method for high-resolution two-dimensional electrophoretic analysis of small (5--10 mg) human muscle biopsy samples with sufficient resolution to resolve the major contractile proteins and enzymes. Using rabbit psoas muscle as a model, we describe methods for sample preparation and two-dimensional electrophoresis. Basic proteins, which appear as streaks when conventional isoelectric focusing is used in the first dimension, are resolved through a modification of the nonequilibrium pH gradient electrophoresis method [Cell 12, 1133 (1977)]. In the two-dimensional patterns obtained from rabbit muscle, we identify the components of 10 enzymes and of myosin, actin, tropomyosin, and troponin. These patterns indicate charge heterogeneity in a large fraction of the proteins. Comparison of rabbit and normal human muscle patterns shows many similarities, but much additional work is required to confirm identifications. We conclude that analysis of small biopsy samples is feasible, but that all aspects of human sample acquisition, storage (when necessary), and preparation require thorough study before the method becomes routine in human muscle research and, ultimately, in the diagnosis of some muscle diseases.

Download Full-text

The oral apparatus of Tetrahymena. V. Oral apparatus polypeptides and their distribution

Journal of Cell Science ◽

10.1242/jcs.44.1.317 ◽

1980 ◽

Vol 44 (1) ◽

pp. 317-333

Author(s):

R.H. Gavin

Keyword(s):

Molecular Weight ◽

Basal Body ◽

Tetrahymena Thermophila ◽

Electrophoretic Analysis ◽

Two Dimensional ◽

Molecular Weight Range ◽

Two Dimensional Electrophoresis ◽

Weight Range ◽

Isoelectric Points ◽

Dimensional Electrophoresis

Two-dimensional electrophoresis was used to resolve approximately 162 polypeptides from the isolated oral apparatus of Tetrahymena thermophila. The molecular weight range was between 110 000 and 15 000 Daltons. The polypeptides had apparent isoelectric points between pH 3.3 and pH 7.2. Electrophoretic analysis of isolated ciliary axonemes and fractionated oral apparatuses made possible the assignment of polypeptides to structures within the oral apparatus. Approximately 24 polypeptides, including alpha and beta tubulins, are probable components of the basal body-basal plate complex. At least 5 of the oral apparatus polypeptides, including alpha and beta tubulin, are components of the oral apparatus ciliary axonemes. Approximately 138 polypeptides are components of the oral apparatus framework.

Download Full-text

Contribution of Sialic Acid to the Heterogeneity of Human Fibrinogen Polypeptide Chains

10.1055/s-0039-1687462 ◽

1979 ◽

Author(s):

C. Kuyas ◽

A. Haeberli ◽

P.W Straub

Keyword(s):

Sialic Acid ◽

Isoelectric Focusing ◽

Acid Content ◽

Polypeptide Chain ◽

Sialic Acid Content ◽

Human Fibrinogen ◽

Single Donor ◽

The Individual ◽

Polypeptide Chains ◽

Vibrio Cholerae Sialidase

Human fibrinogen, isolated from single donor or from pooled plasma, shows a heterogeneity of the Bऔ- and the γ-polypeptide chains on CM-cellulose chromatography. In order to find out whether this heterogeneity is due to the observed differences in sialic acid content of the variants (2 and 1 residue per chain; Gati et al., J.Biol.Chem.253:1315,1978) pooled or single donor fibrinogen (clottability 93-95%) was incubated 24 hrs with either vibrio cholerae sialidase or buffer. The asialofibrinogen (10% of original sialic acid) was compared with intact fibrinogen. After dithiothreitol reduction and alkylation the chains were separated on CM Sepharose. The individual homogeneous Bऔ- and γ-chains were rechromatographed on CM-cellulose. The γ-chain heterogeneity of normal fibrinogen was absent in asialofibrinogen whereas the Bऔ-chain heterogeneity appeared unaffected. Although the variants were indistinguishable on SOS-PAGE, isoelectric focusing in presence of urea demonstrated heterogeneities of both Bऔ- and γ-chains even in asialofibrinogen. Thus, the differences in sialic acid content of the main polypeptide chain variants of pooled as wel l as single donor human fibrinogen can only explain a small part of the polypeptide chain heterogeneity.

Download Full-text