The endogenous vascular elastase that governs development and progression of monocrotaline-induced pulmonary hypertension in rats is a novel enzyme related to the serine proteinase adipsin.
We showed previously a cause and effect relationship between increased activity of an endogenous vascular elastase (EVE) and experimentally induced pulmonary hypertension in rats. We now report the isolation and characterization of EVE. Degenerate oligonucleotides synthesized to homologous sequences in serine elastases were used in a PCR with rat pulmonary artery (PA) cDNA. The PCR product hybridized to a 1.2-kb mRNA and the intensity of hybridization was threefold increased in RNA from rat hypertensive PA at a timepoint when EVE activity was increased. The PCR product was used to screen a cDNA library and sequences obtained encoded rat adipsin. We then used immunoaffinity to purify EVE. An antibody to the elastin-binding protein was used to remove this competitor of elastase from the PA extract and the elastolytic activity increased 100-fold. The enzyme was purified using an antibody that recognizes NH2-terminal sequences of serine proteinases and the eluate was further purified using an antibody raised against recombinant adipsin. A single band at 20 kD immunoreactive with the adipsin antibody was resolved as an active enzyme on an elastin substrate gel. Immunogold labeling with an antibody to an adipsin peptide sequence localized EVE to PA smooth muscle cells. This is the first isolation of EVE; it appears to be a novel enzyme related to the serine proteinase adipsin originally found in adipose tissue.
Zhu
L
L
Division of Cardiovascular Research, Hospital for Sick Children, Toronto, Ontario, Canada.
Wigle
D
D
Hinek
A
A
Kobayashi
J
J
Ye
C
C
Zuker
M
M
Dodo
H
H
Keeley
F W
FW
Rabinovitch
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002417:Cattle; D011416:Complement Factor D; D017931:DNA Primers; D015870:Gene Expression; D006801:Humans; D006976:Hypertension, Pulmonary / Q000139:chemically induced / Q000201:enzymology / Q000503:physiopathology*; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D016686:Monocrotaline; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D010196:Pancreatic Elastase / Q000096:biosynthesis* / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011651:Pulmonary Artery / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry
D017931:DNA Primers; D012333:RNA, Messenger; D016686:Monocrotaline; D012697:Serine Endopeptidases; D010196:Pancreatic Elastase; C493488:CFD protein, human; D011416:Complement Factor D