Purification and IM-Terminal Sequence Analysis of Streptomyces chromofuscus Phospholipase D

1995 ◽  
Vol 107 (1-3) ◽  
pp. 69-71 ◽  
Author(s):  
Tammythan T. Dinh ◽  
David McClure ◽  
Donald A. Kennerly
Keyword(s):  
Sequence Analysis ◽  
Phospholipase D ◽  
Terminal Sequence ◽  
Streptomyces Chromofuscus

The alkylated thiohydantoin method for C-terminal sequence analysis

2000 ◽  
pp. 119-131
Author(s):  
David R. Dupont ◽  
MeriLisa Bozzini ◽  
Victoria L. Boyd
Keyword(s):  
Sequence Analysis ◽  
Terminal Sequence

scholarly journals Transphosphatidylation capacity of phospholipase D from cabbage (Brassica oleracea L. var. capitata L.) leaves and Streptomyces chromofuscus

1999 ◽  
Vol 25 (5) ◽  
pp. 229-237
Author(s):  
Toshihiro WATANABE ◽  
Hiroaki SATO ◽  
Yozo NAKAZAWA ◽  
Yoshimasa SAGANE ◽  
T. Tsuneo KOZIMA ◽  
...  
Keyword(s):  
Brassica Oleracea ◽  
Phospholipase D ◽  
Streptomyces Chromofuscus ◽  
Brassica Oleracea L

Automated Carboxy-Terminal Sequence Analysis of Polypeptides Containing C-Terminal Proline

1995 ◽  
Vol 224 (2) ◽  
pp. 588-596 ◽  
Author(s):  
J.M. Bailey ◽  
O. Tu ◽  
G. Issai ◽  
A. Ha ◽  
J.E. Shively
Keyword(s):  
Sequence Analysis ◽  
Terminal Sequence ◽  
Carboxy Terminal

scholarly journals Human Neutrophil Elastase Activates Human Factor V but Inactivates Thrombin-Activated Human Factor V

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 1065-1074 ◽  
Author(s):  
John A. Samis ◽  
Marilyn Garrett ◽  
Reginald P. Manuel ◽  
Michael E. Nesheim ◽  
Alan R. Giles
Keyword(s):  
Sequence Analysis ◽  
Heavy Chain ◽  
Human Neutrophil ◽  
Human Factor ◽  
Time Dependent ◽  
Human Neutrophil Elastase ◽  
Factor V ◽  
Terminal Sequence ◽  
Sds Page ◽  
Cofactor Activity

The effect of human neutrophil elastase (HNE) on human factor V (F.V) or α-thrombin–activated human factor V (F.Va) was studied in vitro by prothrombinase assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and NH2 -terminal sequence analysis. Incubation of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in a time-dependent increase in its cofactor activity. In contrast, treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted only in a time-dependent decrease in its cofactor activity. Under the conditions of these experiments, the maximum extent of F.V activation accomplished by incubation with HNE was approximately 65% to 70% of that observed with α-thrombin in presence of Ca2+. The extent of both the HNE-dependent enhancement in F.V cofactor activity and the HNE-dependent decrease in F.Va cofactor activity was not influenced by the addition of phosphatidylcholine/phosphatidylserine (PCPS) vesicles (50 μmol/L). The HNE-derived cleavage products of F.V, which correlated with increased cofactor activity, as demonstrated by SDS-PAGE under reducing conditions, were different from those generated using α-thrombin. Treatment of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in the production of three closely spaced doublets of: 99/97, 89/87, and 76/74 kD whose appearance over time correlated well with the increased cofactor activity as judged by densitometry. Treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted in the cleavage of both the 96 kD heavy chain and the 74/72 kD light chain into products of: 56, 53, 35, 28, 22, and 12 kD. Although densitometry indicated that both the heavy and light chains of F.Va were hydrolyzed by HNE, cleavage of the 96 kD heavy chain was more extensive during the time period (10 to 30 minutes) of the greatest loss of F.Va cofactor activity. NH2 -terminal sequence analysis of F.V treated with HNE indicated cleavage at Ile819 and Ile1484 under conditions during which the procofactor expressed enhanced cofactor activity in the prothrombinase complex. NH2 -terminal sequence analysis of F.Va treated with HNE indicated cleavage at Ala341, Ile508, and Thr1767 under conditions, which the cofactor became inactivated, as measured by prothrombinase activity. The activation and inactivation cleavage sites are close to those cleaved by the physiological activator and inactivator of F.V and F.Va, namely α-thrombin (Arg709 and Arg1545) and Activated Protein C (APC) (Arg306 and Arg506), respectively. These results indicate that HNE can generate proteolytic products of F.V, which initially express significantly enhanced procoagulant cofactor activity similar to that observed following activation with α-thrombin. In contrast, HNE treatment of F.Va resulted only in the loss of its cofactor activity, but again, this is similar to that observed following inactivation by APC.

Prothrombin Fragment 1.2.3, A Major Product Of Prothrombin Activation In Human Plasma

1981 ◽  
Author(s):  
M J Rabiet ◽  
B Furie ◽  
B C Furie
Keyword(s):  
Sequence Analysis ◽  
Human Plasma ◽  
Gel Electrophoresis ◽  
Factor Xa ◽  
Normal Human Plasma ◽  
Terminal Sequence ◽  
Amino Terminal ◽  
Normal Human ◽  
Amino Terminal Sequence ◽  
Prothrombin Activation

The conversion of human prothrombin to thrombin is associated with a number of cleavage intermediates and products whose appearance and concentration are dependent upon the prothrombin activation conditions used. In the current investigation, the fragments of prothrombin which appear in normal human plasma after activation of the blood coagulation cascade were studied. Radioiodinated human prothrombin was added to platelet-poor relipidated normal human plasma and clotting initiated with Ca(II) and kaolin. The radiolabeled prothrombin cleavage products which formed were analyzed by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate (SDS) and 2-mercaptoethanol (2-ME), A new product of prothrombin activation was observed. Its migration was more rapid than prethrombin 1 and slower than fragment 1.2. No previously identified products of prothrombin activation migrated to the same position in the gel.The previously unrecognized fragment was identified as fragment 1.2.3 as follows. Prothrombin was activated by factor Xa in the presence of Ca(II) and phospholipid. The desired product was isolated by absorption to and elution from barium citrate and by DEAE cellulose chromatography. This purified material, migrating identically with the unknown plasma product was homogeneous upon SDS gel electrophoresis with 2-ME. The amino terminal sequence of the isolated material was identical to that of prothrombin. Digestion of this material with either factor Xa or thrombin yielded as major products fragment 1.2 and fragment 1. (Fragment 2 and fragment 3 eluted from the gels under the conditions employed). Amino terminal sequence analysis of the factor Xa digestion products of the isolated material indicated three amino acid residues at each cycle. The sequences of fragment 1, fragment 2, and fragment 3 are consistent with this sequence analysis. On this basis we suggest that fragment 1.2.3 is a prominent product of prothrombin conversion to thrombin in plasma.

scholarly journals Symmetrically substituted dichlorophenes inhibit N-acyl-phosphatidylethanolamine phospholipase D

2020 ◽  
Vol 295 (21) ◽  
pp. 7289-7300 ◽  
Author(s):  
Geetika Aggarwal ◽  
Jonah E. Zarrow ◽  
Zahra Mashhadi ◽  
C. Robb Flynn ◽  
Paige Vinson ◽  
...  
Keyword(s):  
Bile Acids ◽  
Phospholipase D ◽  
Cultured Cells ◽  
Lithocholic Acid ◽  
Hek293 Cells ◽  
Serum Lipase ◽  
Structure Activity ◽  
Molecule Inhibitor ◽  
Streptomyces Chromofuscus

N-Acyl-phosphatidylethanolamine phospholipase D (NAPE-PLD) (EC 3.1.4.4) catalyzes the final step in the biosynthesis of N-acyl-ethanolamides. Reduced NAPE-PLD expression and activity may contribute to obesity and inflammation, but a lack of effective NAPE-PLD inhibitors has been a major obstacle to elucidating the role of NAPE-PLD and N-acyl-ethanolamide biosynthesis in these processes. The endogenous bile acid lithocholic acid (LCA) inhibits NAPE-PLD activity (with an IC50 of 68 μm), but LCA is also a highly potent ligand for TGR5 (EC50 0.52 μm). Recently, the first selective small-molecule inhibitor of NAPE-PLD, ARN19874, has been reported (having an IC50 of 34 μm). To identify more potent inhibitors of NAPE-PLD, here we used a quenched fluorescent NAPE analog, PED-A1, as a substrate for recombinant mouse Nape-pld to screen a panel of bile acids and a library of experimental compounds (the Spectrum Collection). Muricholic acids and several other bile acids inhibited Nape-pld with potency similar to that of LCA. We identified 14 potent Nape-pld inhibitors in the Spectrum Collection, with the two most potent (IC50 = ∼2 μm) being symmetrically substituted dichlorophenes, i.e. hexachlorophene and bithionol. Structure–activity relationship assays using additional substituted dichlorophenes identified key moieties needed for Nape-pld inhibition. Both hexachlorophene and bithionol exhibited significant selectivity for Nape-pld compared with nontarget lipase activities such as Streptomyces chromofuscus PLD or serum lipase. Both also effectively inhibited NAPE-PLD activity in cultured HEK293 cells. We conclude that symmetrically substituted dichlorophenes potently inhibit NAPE-PLD in cultured cells and have significant selectivity for NAPE-PLD versus other tissue-associated lipases.

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