scholarly journals Expression in a cell-free system of normal and variant forms of human antithrombin III. Ability to bind heparin and react with alpha-thrombin

Blood ◽  
1990 ◽  
Vol 76 (8) ◽  
pp. 1521-1529 ◽  
Author(s):  
RC Austin ◽  
RA Rachubinski ◽  
F Fernandez-Rachubinski ◽  
MA Blajchman
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Translation Product ◽  
Free System ◽  
Reactive Center ◽  
At Iii ◽  
Free Translation

Abstract Human antithrombin III (AT-III) cDNA was cloned into the cell-free expression phagemid vector pGEM-3Zf(+) and site-directed mutagenesis was used to remove nucleotides encoding the signal peptide. AT-III messenger RNA (mRNA) transcripts derived from this construct were translated in an mRNA-dependent rabbit reticulocyte lysate (RRL) system containing (35S)methionine. Immunoprecipitation of the cell-free translation mixture with rabbit polyclonal antibodies to AT-III showed, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE), a 47-Kd polypeptide which is the non-glycosylated mature form of plasma AT-III. Densitometric scanning showed that this polypeptide constitutes greater than 90% of the radiolabeled polypeptides produced in this system. Heparin-Sepharose chromatography resulted in the elution of cell-free derived AT-III as a broad peak between 0.2 and 0.7 mol/L NaCl. The cell-free derived AT-III also reacted with human alpha- thrombin. In 2 minutes approximately 20% of the AT-III was found associated with a higher molecular weight species, consistent with the formation of a 1:1 stoichiometric covalent complex between alpha- thrombin and AT-III. Unfractionated heparin accelerated the rate of formation of such complexes. When Ser394 was mutated to Leu to form the AT-III Denver mutant, the cell-free translation product of this mutation did not show any significant complex formation when reacted with alpha-thrombin. A truncated form of AT-III (Met251-Lys432), containing only the putative thrombin-binding domain, was synthesized independently. This 21-Kd polypeptide did not bind heparin; however, it was cleaved by alpha-thrombin presumably at the reactive center Arg393- Ser394. When Ser394 was mutated to Leu the cell-free translation product of this truncated AT-III mutation did not react with alpha- thrombin at the reactive center. This simple cell-free approach, along with site-directed mutagenesis, should allow for the rapid and accurate mapping of the functional domains of human AT-III.

scholarly journals Expression in a cell-free system of normal and variant forms of human antithrombin III. Ability to bind heparin and react with alpha-thrombin

Blood ◽  
1990 ◽  
Vol 76 (8) ◽  
pp. 1521-1529
Author(s):  
RC Austin ◽  
RA Rachubinski ◽  
F Fernandez-Rachubinski ◽  
MA Blajchman
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Translation Product ◽  
Free System ◽  
Reactive Center ◽  
At Iii ◽  
Free Translation

Human antithrombin III (AT-III) cDNA was cloned into the cell-free expression phagemid vector pGEM-3Zf(+) and site-directed mutagenesis was used to remove nucleotides encoding the signal peptide. AT-III messenger RNA (mRNA) transcripts derived from this construct were translated in an mRNA-dependent rabbit reticulocyte lysate (RRL) system containing (35S)methionine. Immunoprecipitation of the cell-free translation mixture with rabbit polyclonal antibodies to AT-III showed, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE), a 47-Kd polypeptide which is the non-glycosylated mature form of plasma AT-III. Densitometric scanning showed that this polypeptide constitutes greater than 90% of the radiolabeled polypeptides produced in this system. Heparin-Sepharose chromatography resulted in the elution of cell-free derived AT-III as a broad peak between 0.2 and 0.7 mol/L NaCl. The cell-free derived AT-III also reacted with human alpha- thrombin. In 2 minutes approximately 20% of the AT-III was found associated with a higher molecular weight species, consistent with the formation of a 1:1 stoichiometric covalent complex between alpha- thrombin and AT-III. Unfractionated heparin accelerated the rate of formation of such complexes. When Ser394 was mutated to Leu to form the AT-III Denver mutant, the cell-free translation product of this mutation did not show any significant complex formation when reacted with alpha-thrombin. A truncated form of AT-III (Met251-Lys432), containing only the putative thrombin-binding domain, was synthesized independently. This 21-Kd polypeptide did not bind heparin; however, it was cleaved by alpha-thrombin presumably at the reactive center Arg393- Ser394. When Ser394 was mutated to Leu the cell-free translation product of this truncated AT-III mutation did not react with alpha- thrombin at the reactive center. This simple cell-free approach, along with site-directed mutagenesis, should allow for the rapid and accurate mapping of the functional domains of human AT-III.

scholarly journals Purified radiolabeled antithrombin III metabolism in three families with hereditary AT III deficiency: application of a three-compartment model

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 93-98
Author(s):  
EA Knot ◽  
E de Jong ◽  
JW ten Cate ◽  
AH Iburg ◽  
CP Henny ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Compartment Model ◽  
Control Group ◽  
Surface Binding ◽  
Catabolic Rate ◽  
At Iii ◽  
Three Compartment Model ◽  
The Mean

Purified human radioiodinated antithrombin III (125I-AT III) was used to study its metabolism in six members from three different families with a known hereditary AT III deficiency. Six healthy volunteers served as a control group. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and crossed immunoelectrophoresis (CIE) showed the purified AT III to be homogeneous. Amino acid analysis of the protein revealed a composition identical to a highly purified internal standard. The specific activity was 5.6 U/mg. Analysis of plasma radioactivity data was performed, using a three-compartment model. Neither plasma disappearance half-times nor fractional catabolic rate constants differed significantly between patients and control subjects. The mean absolute catabolic rate in the patient group was significantly lower than that of the control group at 2.57 +/- 0.44 and 4.46 +/- 0.80 mg/kg/day, respectively. In addition, the mean patient alpha 1-phase, flux ratio (k1,2 and k2,1) of the second compartment alpha 2-phase and influx (k3,1) of the third compartment were significantly reduced as compared with control values. It has been tentatively concluded that the observed reduction in the second compartment may be caused by a decrease in endothelial cell surface binding.

scholarly journals Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 496-500 ◽  
Author(s):  
M Wolf ◽  
C Boyer ◽  
A Tripodi ◽  
D Meyer ◽  
MJ Larrieu ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Two Dimensional ◽  
Western Blots ◽  
Sds Page ◽  
New Variant ◽  
At Iii ◽  
Monospecific Antisera

Abstract A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.

scholarly journals Antithrombin-III-Hamilton, Ala 382 to Thr: an antithrombin-III variant that acts as a substrate but not an inhibitor of alpha-thrombin and factor Xa

Blood ◽  
1991 ◽  
Vol 77 (10) ◽  
pp. 2185-2189
Author(s):  
RC Austin ◽  
RA Rachubinski ◽  
FA Ofosu ◽  
MA Blajchman
Keyword(s):  
Complex Formation ◽  
Serine Proteases ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Biological Behavior ◽  
Form Complex ◽  
Free System ◽  
Reactive Center ◽  
Sds Page ◽  
At Iii

Antithrombin-III-Hamilton has been shown to be a structural variant of antithrombin-III (AT-III) with normal heparin affinity but impaired protease inhibitory activity. The molecular defect of AT-III-Hamilton is the substitution of Thr for Ala at amino acid residue 382. The plasma of affected individuals contains approximately equal quantities of normal AT-III and AT-III-Hamilton. When AT-III was isolated from the plasma of the propositus by heparin-Sepharose chromatography, it had identical mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to normal plasma-derived AT-III, under both reducing and nonreducing conditions. However, the AT-III-Hamilton species, separated from the propositus' normal AT-III by a combination of heparin-Sepharose and thrombin-Sepharose chromatography, had increased mobility on reductive SDS-PAGE compared with AT-III from the propositus isolated by heparin-Sepharose chromatography alone. Under nonreducing conditions this AT-III-Hamilton species had decreased mobility compared with AT-III from the propositus (or normal AT-III) isolated only by heparin-Sepharose chromatography. When incubated with either human alpha-thrombin or human factor Xa, this AT-III-Hamilton species was unreactive. Approximately 50% of the AT-III from the propositus isolated by heparin-Sepharose chromatography, when incubated with either human alpha-thrombin or factor Xa, did not form complex but was cleaved, presumably at the reactive center Arg393-Ser394. To further substantiate the biological behavior of this variant, AT-III- Hamilton polypeptides were synthesized in a cell-free system. This recombinantly produced AT-III-Hamilton, when incubated with either human alpha-thrombin or factor Xa, was cleaved by both these proteases, but did not show any complex formation. The results indicate that AT- III-Hamilton does not form a stable covalent inhibitory complex with these serine proteases but can be cleaved at the reactive center. Thus, the inhibition of serine proteases by their natural inhibitors (the serpins) involves at least two separate, but interrelated events; hydrolysis at the reactive center followed by complex formation. AT-III- Hamilton is capable of only the first of these events.

scholarly journals Antithrombin-III-Hamilton, Ala 382 to Thr: an antithrombin-III variant that acts as a substrate but not an inhibitor of alpha-thrombin and factor Xa

Blood ◽  
1991 ◽  
Vol 77 (10) ◽  
pp. 2185-2189 ◽  
Author(s):  
RC Austin ◽  
RA Rachubinski ◽  
FA Ofosu ◽  
MA Blajchman
Keyword(s):  
Complex Formation ◽  
Serine Proteases ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Biological Behavior ◽  
Form Complex ◽  
Free System ◽  
Reactive Center ◽  
Sds Page ◽  
At Iii

Abstract Antithrombin-III-Hamilton has been shown to be a structural variant of antithrombin-III (AT-III) with normal heparin affinity but impaired protease inhibitory activity. The molecular defect of AT-III-Hamilton is the substitution of Thr for Ala at amino acid residue 382. The plasma of affected individuals contains approximately equal quantities of normal AT-III and AT-III-Hamilton. When AT-III was isolated from the plasma of the propositus by heparin-Sepharose chromatography, it had identical mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to normal plasma-derived AT-III, under both reducing and nonreducing conditions. However, the AT-III-Hamilton species, separated from the propositus' normal AT-III by a combination of heparin-Sepharose and thrombin-Sepharose chromatography, had increased mobility on reductive SDS-PAGE compared with AT-III from the propositus isolated by heparin-Sepharose chromatography alone. Under nonreducing conditions this AT-III-Hamilton species had decreased mobility compared with AT-III from the propositus (or normal AT-III) isolated only by heparin-Sepharose chromatography. When incubated with either human alpha-thrombin or human factor Xa, this AT-III-Hamilton species was unreactive. Approximately 50% of the AT-III from the propositus isolated by heparin-Sepharose chromatography, when incubated with either human alpha-thrombin or factor Xa, did not form complex but was cleaved, presumably at the reactive center Arg393-Ser394. To further substantiate the biological behavior of this variant, AT-III- Hamilton polypeptides were synthesized in a cell-free system. This recombinantly produced AT-III-Hamilton, when incubated with either human alpha-thrombin or factor Xa, was cleaved by both these proteases, but did not show any complex formation. The results indicate that AT- III-Hamilton does not form a stable covalent inhibitory complex with these serine proteases but can be cleaved at the reactive center. Thus, the inhibition of serine proteases by their natural inhibitors (the serpins) involves at least two separate, but interrelated events; hydrolysis at the reactive center followed by complex formation. AT-III- Hamilton is capable of only the first of these events.

scholarly journals Iodine-mediated inactivation of lipid- and nonlipid-enveloped viruses in human antithrombin III concentrate

Blood ◽  
1995 ◽  
Vol 86 (2) ◽  
pp. 791-796 ◽  
Author(s):  
F Highsmith ◽  
H Xue ◽  
X Chen ◽  
L Benade ◽  
J Owens ◽  
...  
Keyword(s):  
Sindbis Virus ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Albumin ◽  
Enveloped Viruses ◽  
Complete Inactivation ◽  
Ensure Patient Safety ◽  
Sds Page ◽  
At Iii

Human plasma-derived protein concentrates intended for clinical use must be treated for viral inactivation to ensure patient safety. This study explored the use of liquid iodine for inactivation of several lipid- and nonlipid-enveloped viruses in an antithrombin III (AT-III) concentrate. Iodine at levels of 0.01% to 0.02% caused between 43% and 94% loss of AT-III activity, as well as degradation of AT-III as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. However, addition of up to 0.1% human albumin protected the AT-III against both inactivation and fragmentation. At albumin levels sufficient to retain greater than 75% of AT-III activity, greater than 6 logs of sindbis, encephalomyocarditis, and vesicular stomatitis viruses, greater than 4 logs of pseudorabies, and greater than 3 logs of human immunodeficiency virus were inactivated. Except with sindbis virus, this represented complete inactivation of all the viruses spiked into the AT-III concentrate.

Wheat embryo ribonucleates XI. Conserved mRNA in dry wheat embryos and its relation to protein synthesis during early imbibition

1978 ◽  
Vol 56 (6) ◽  
pp. 365-369 ◽  
Author(s):  
A. C. Cuming ◽  
B. G. Lane
Keyword(s):  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Free System ◽  
Wheat Embryo ◽  
Free Translation ◽  
Wheat Embryos ◽  
The Many

It has been found that bulk poiy(A)-rich RNA from dry wheat embryos is broadly hetero-disperse when examined by polyacrylamide gel electrophoresis. The poly(A)-rich RNA from dry wheat embryos has been translated in a cell-free protein-synthesizing system from the same commerically supplied, roller-milled wheat embryos. Compatible with the electrophoretic heterodispersity observed for poly(A)-rich RNA, the radioactive products of its cell-free translation, when examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis, have mobilities that are broadly coincident with the many dye-stained (nonradioactive) proteins present in wheat extracts. With due allowance for the limitations of the cell-free system, which is known to translate, selectively, lower molecular-weight species of mRNA, it has been concluded that the conserved poly(A)-rich mRNA in dry wheat embryos probably has the translational capacity required to account for the highly eclectic protein synthesis that we have observed during early (40-min) imbibition of viable wheat embryos.

scholarly journals Synthesis of 3-ketoacyl-CoA thiolase of rat liver peroxisomes on free polyribosomes as a larger precursor. Induction of thiolase mRNA activity by clofibrate

1985 ◽  
Vol 226 (3) ◽  
pp. 697-704 ◽  
Author(s):  
Y Fujiki ◽  
R A Rachubinski ◽  
R M Mortensen ◽  
P B Lazarow
Keyword(s):  
Rat Liver ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Immunoblot Analysis ◽  
Polyacrylamide Gel ◽  
Proteolytic Processing ◽  
Translation Product ◽  
Free Translation

The site of synthesis and induction by clofibrate of peroxisomal 3-ketoacyl-CoA thiolase (acetyl-CoA acyltransferase; EC 2.3.1.16) was investigated. Free and membrane-bound polyribosomal RNA species from the livers of normal rats and rats treated with clofibrate, a hypolipidaemic drug that causes marked proliferation of peroxisomes, were translated in a nuclease-treated rabbit reticulocyte-lysate cell-free protein-synthesizing system with [35S]methionine as label. The cell-free translation products were immunoprecipitated with monospecific X rabbit anti-thiolase serum and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and fluorography. Thiolase mRNA was found predominantly in free polyribosomes, in both normal and clofibrate-treated rats. Clofibrate treatment increased mRNA activity for thiolase approx. 20-fold. The translation product of clofibrate-induced thiolase mRNA migrated slightly faster in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis than did the translation product of normal thiolase mRNA. Both the normal and the clofibrate-induced translation products were approx. 6000 Da larger than the 41000-Da subunit of the purified enzyme. Immunoblot analysis of liver homogenates, isolated peroxisomes and the purified enzyme indicated that the thiolase subunit was approx. 41000 Da in all samples, ruling out proteolysis during the purification of thiolase. Thiolase biogenesis thus differs from that of rat liver peroxisomal proteins studied previously in that it is synthesized as a larger precursor, implying post-translational import of thiolase into peroxisomes with proteolytic processing. Clofibrate apparently alters the size as well as the amount of the translation product.

scholarly journals Purified radiolabeled antithrombin III metabolism in three families with hereditary AT III deficiency: application of a three-compartment model

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 93-98 ◽  
Author(s):  
EA Knot ◽  
E de Jong ◽  
JW ten Cate ◽  
AH Iburg ◽  
CP Henny ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Compartment Model ◽  
Control Group ◽  
Surface Binding ◽  
Catabolic Rate ◽  
At Iii ◽  
Three Compartment Model ◽  
The Mean

Abstract Purified human radioiodinated antithrombin III (125I-AT III) was used to study its metabolism in six members from three different families with a known hereditary AT III deficiency. Six healthy volunteers served as a control group. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and crossed immunoelectrophoresis (CIE) showed the purified AT III to be homogeneous. Amino acid analysis of the protein revealed a composition identical to a highly purified internal standard. The specific activity was 5.6 U/mg. Analysis of plasma radioactivity data was performed, using a three-compartment model. Neither plasma disappearance half-times nor fractional catabolic rate constants differed significantly between patients and control subjects. The mean absolute catabolic rate in the patient group was significantly lower than that of the control group at 2.57 +/- 0.44 and 4.46 +/- 0.80 mg/kg/day, respectively. In addition, the mean patient alpha 1-phase, flux ratio (k1,2 and k2,1) of the second compartment alpha 2-phase and influx (k3,1) of the third compartment were significantly reduced as compared with control values. It has been tentatively concluded that the observed reduction in the second compartment may be caused by a decrease in endothelial cell surface binding.

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