Abstract
We have compared the cleavage of purified human Hageman factor (HF) by an activated form of human Hageman factor (HFa) (autodigestion) and by kallikrein. In each case, an initial cleavage is seen which produces HFa with Mr = 80,000 consisting of a heavy chain of Mr = 52,000 disulfide-linked to a light chain of Mr = 28,000. As autodigestion proceeds, HFa is shown to be further digested to yield a major active product at a molecular weight of 40,000 as well as Hageman factor fragment (HFf), which appear as two closely related molecular species of Mr = 28,000 and 30,000. A minor active product of Mr = 70,000 is also seen. Upon reduction of each of the active forms, a chain with Mr = 28,000 is released which contains the active site. HF digestion by kallikrein results in rapid formation of HFa, followed by HFa digestion to HFf and degradation of the heavy chain region to an inactive fragment at 40,000 daltons, which is then degraded to an end product of Mr = 36,000. Production of the active species with Mr = 40,000 and 70,000 is greatly diminished when kallikrein is the HF activator, and these active forms are shown to be formed primarily by autodigestion. The time course of HFa and HFf formation indicates that the rate of activation of Hageman factor by kallikrein is much faster than the rate of autoactivation; the addition of high molecular weight kininogen increases the rate of HFa and HFf formation as well as the extent of HG digestion. These data indicate that HFa is the active intermediate from which other active species are derived. The patterns of HF and HFa digestion by HFa and kallikrein are distinct; a model for HF digestion is presented.
Highlights
Hageman factor (HFa) and by kallik- a slow autodigestion and autoactivation of native surfacerein
The cleavage of native HF toform HFa involves digestion within a disulfide bridge to form a two-chain activated enzyme in which the active site is in the light chain [11].When Rev& et al studied the activation of HF upon surfaces ( l l ), only HFa and HFf formation were demonstrated, fluid phase activation generated one of the intermediates[12]
40,000 andas a lightchaindoublet simultaneously. This of H F which show the presence of active sites(asindicated by demonstrates that during autoactivation there is an initial the uptakeof the DNS-GGACMK) contain a disulfide-linked cleavage resulting in the formation of the two-chain disulfide- light chain with a'molecular weight of 28,000, purified H F was linked HFa which is followed by either of two alternative allowed to autodigest on kaolin and was labeled with cleavages
Summary
Protein concentrationswere determined using the methodof Lowry for 9 h. The mixture was addedto apolypropylene standard was determinedspectrophotometrically using thevalue Microfuge tube in which Bolton-Hunter reagent (250 pCi) had been. Purified H F was radiolabeledby placing "'I-Bolton-Hunter reagent SDS. The gels were removed from the tubes and placed onto overnight a t room temperature, and the"'I-HF was dialyzed exhaus- 12%S D S slab gels with a stacking layerof 1%agarose in 0.12 M Tristively in 10 mM Napi, pH7.8, made 10 mM in benzamidine. HFa (56 pg/ml) was radiolabeled by the lactoperoxidase method Two wells wereformed in the stacking agarose, and the tube gels [18] using "'I-sodium iodide in 0.12 M Nap, buffer, pH 7.4, containing were embedded between the wells by overlaying them with hot.
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