Cytosolic phospholipase A2 initiates the biosynthesis of prostaglandins, leukotrienes, and platelet-activating factor (PAF), mediators of the pathophysiology of asthma and arthritis. Here, we report the X-ray crystal structure of human cPLA2 at 2.5 A. cPLA2 consists of an N-terminal calcium-dependent lipid-binding/C2 domain and a catalytic unit whose topology is distinct from that of other lipases. An unusual Ser-Asp dyad located in a deep cleft at the center of a predominantly hydrophobic funnel selectively cleaves arachidonyl phospholipids. The structure reveals a flexible lid that must move to allow substrate access to the active site, thus explaining the interfacial activation of this important lipase.
The X-ray structure of GMD reveals that it is a member of the short-chain dehydrogenase/reductase (SDR) family of proteins. We have modeled the binding of NADP and GDP-mannose to the enzyme and mutated four of the active-site residues to determine their function. The combined modeling and mutagenesis data suggests that at position 133 threonine substitutes serine as part of the serine-tyrosine-lysine catalytic triad common to the SDR family and Glu 135 functions as an active-site base.
Es wird die Reinigung von Phospholipase A aus menschlichem,
mittels Duodenalsondierung gewonnenem, Pankreassekret beschrieben. Das Enzym
liegt hier in wesentlich höherer spezifischer Aktivität vor als im Pankreas. Das
Reinigungsverfahren ist relativ einfach und führt zu einem elektrophoretisch einheitlichen
Enzympräparat. Zur Reinigung werden Proteinfällungen in Hitze, mit
Aceton, Säure und Ammonsulfat durchgeführt. Es folgen Gelfiltration an Sephadex
G 50 oder G 75 und Zelluloseacetat-Elektrophorese. Bei einer Aktivitätsausbeute
von 10—15% wird eine ca. 100fache Reinigung des Enzyms erzielt.
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