A simple two-step purification of protease nexin.

This paper describes a simple purification procedure for protease nexin, a serine proteinase inhibitor secreted by cultured human fibroblasts that regulates proteinase activity at and near the cell surface. The first step in the procedure takes advantage of the high-affinity binding of protease nexin to dextran sulphate-Sepharose. This step eliminates the need for prior concentration of the serum-free fibroblast-conditioned medium, since protease nexin binds to the resin in the presence of physiological saline. The use of dextran sulphate also provides an affinity resin with considerably less variability than the heparin-based resins previously used. Final purification to homogeneity involves a combination of DEAE-Sepharose in-line with dextran sulphate-Sepharose to simultaneously purify and concentrate the protein. Purified protease nexin is shown by Ouchterlony analysis and peptide mapping to be immunologically and structurally distinct from antithrombin III and heparin cofactor II, two plasma proteinase inhibitors with similar properties.     

A modified procedure for the purification of clostridial collagenase.

A method is described for the purification of clostridial collagenase from a crude enzyme preparation employing cation exchange chromatography on SP Sephadex, anion exchange chromatography on DEAE cellulose and gel filtration on Sephacryl S-200. Emphasis was placed on purity using continuous shallow gradients for the ion exchange separations to increase resolution and monitoring eluates both with respect to ultraviolet light absorption at 230 nm and analytical disc gel acrylamide electrophoresis. In addition, protein fractions were assayed for collagenolytic and non-specific proteolytic activity. The purity of the final preparation was assessed by acrylamide electrophoresis, gel filtration and amino acid analysis. The isolated enzyme hydrolyzed between 30 and 40% of rat tail tendon collagen in 1 h at 37 degrees C and lacked measurable trypsin or elastase-like activity.     

Protease specificity and heparin binding and activation of recombinant protease nexin I

Structural and functional properties of alpha-protease nexin I (alpha-PNI) expressed in Chinese hamster ovary cells were studied. All three cysteines were in the reduced form, showing that the potential disulfide bridge between residues Cys117 and Cys131 was not formed. Heparin association rate enhancements were from ka = 8.3 x 10(5) to 0.7-1.6 x 10(9) M-1 s-1 for the interaction of PNI with thrombin, from ka = 5.1 x 10(3) to 3.5 x 10(5) M-1 s-1 for interaction with Factor Xa, and from ka = 2.2 x 10(6) to 1.0 x 10(7) M-1 s-1 for interaction with trypsin; there was no rate enhancement of the plasmin interaction (ka = 1.0 x 10(5) M-1 s-1). The minimal heparin pentasaccharide had no effect on these interactions. Cleavage of the reactive center loop of PNI by three different proteases gave the typical stressed to relaxed change in thermal stability, but unlike with antithrombin III, there was no loss of heparin affinity. A similar difference from antithrombin was that PNI-thrombin complexes retained normal heparin affinity. These results are compatible with a role for protease nexin I as a cell-associated thrombin inhibitor that remains bound to the cell surface even after complexing with the protease, as compared with the role of antithrombin III as a circulating inhibitor of thrombin that becomes activated on binding to the microvasculature and is released on complex formation.     

Protease nexin 1 is a potent urinary plasminogen activator inhibitor in the presence of collagen type IV

Protease nexin 1 (PN1) in solution forms inhibitory complexes with thrombin or urokinase, which have opposing effects on the blood coagulation cascade. An initial report provided data supporting the idea that PN1 target protease specificity is under the influence of collagen type IV (1). Although collagen type IV demonstrated no effect on the association rate between PN1 and thrombin, the study reported that the association rate between PN1 and urokinase was allosterically reduced 10-fold. This has led to the generally accepted idea that the primary role of PN1 in the brain is to act as a rapid thrombin inhibition and clearance mechanism during trauma and loss of vascular integrity. In studies to identify the structural determinants of PN1 that mediate the allosteric interaction with collagen type IV, we found that protease specificity was only affected after transient exposure of PN1 to acidic conditions that mimic the elution protocol from a monoclonal antibody column. Because PN1 used in previous studies was purified over a monoclonal antibody column, we propose that the allosteric regulation of PN1 target protease specificity by collagen type IV is a result of the purification protocol. We provide both biochemical and kinetic data to support this conclusion. This finding is significant because it implies that PN1 may play a much larger role in the modeling and remodeling of brain tissues during development and is not simply an extravasated thrombin clearance mechanism as previously suggested.     

A simple procedure for tenascin purification.

Here we describe a two-step procedure for purification of human tenascin from conditioned medium of the SK-MEL-28 human melanoma cell line. The first step consists in passing the conditioned media through two chromatography columns connected in sequence. The first is a large capacity gelatin--Sepharose affinity chromatography column (to remove fibronectin), the second, over which the unbound material from the first column flows directly, is a hydroxyapatite chromatography column. Under these conditions, all tenascin present in the conditioned medium binds to the hydroxyapatite chromatography column from which it is then eluted by a 5-300 mM sodium phosphate gradient. With this step, we obtain a crude tenascin preparation, concentrated about 20 times with respect to the starting conditioned medium, and in which tenascin represents more than 50% of the total protein. The second step consists of two sequential precipitations with 6% and 12.8% poly(ethylene glycol). After this step, tenascin is more than 95% pure and does not show any contamination of chondroitin-sulfate-containing proteoglycans that are known to bind to it. From 21 medium we obtain about 3-4 mg tenascin which corresponds to a yield of about 40-50%. This procedure gives a higher yield, is simpler with respect to procedures previously described, avoids the exposure of the protein to denaturing agents or harsh conditions and could be used for purification of tenascin from the conditioned media of other cell lines. Thus, this procedure may represent a simple and useful tool for the preparation of tenascin to study its biological functions.     

A modified mammalian tandem affinity purification procedure to prepare functional polycystin-2 channel

The tandem affinity purification (TAP) procedure was initially developed as a tool for rapid purification of native protein complexes expressed at their natural levels in yeast cells. This purification procedure was also applied to study interactions between soluble proteins in mammalian cells. In order to apply this procedure to mammalian membrane proteins, we created a modified TAP tag expression vector and fused with the PKD2 gene, encoding a membrane cation channel protein, polycystin-2, mutated in 15% of autosomal dominant polycystic kidney disease. We generated epithelial Madin-Darby canine kidney cell line stably expressing TAP-tagged polycystin-2, improved the subsequent steps for membrane protein release and stability, and succeeded in purifying this protein. Using patch clamp electrophysiology, we detected specific polycystin-2 channel activities when the purified protein was reconstituted into a lipid bilayer system. Thus, this modified TAP procedure provides a powerful alternative to functionally characterize membrane proteins, such as ion channels, transporters and receptors, using cell-free system derived from mammalian cells.     

Protease inhibitors from broad bean isolation and purification

Four protease inhibitors were demonstrated and two, BBPI-1 and BBPI-2, were purified from broad bean seeds using a combination of (NH₄)₂SO₄ fractionation, ion-exchange chromatography on CM 52-cellulose and CM 50 Sephadex. BBPI-1 and 2 had broad specificity and inhibited trypsin, chymotrypsin, thrombin, pronase and papain. Both inhibitors were heat stable, had a wide pH tolerance, a MW of approximately 11 000 and contained 14·5% N. BBPI-1 and 2 had a pI of 8·5 and 7·5 respectively.     

A simple purification procedure for monomeric nucleosomes.

The soluble chromatin fragments from nuclei of avian erythrocytes digested with micrococcal nuclease were fractionated by the addition of sodium phosphate to 0.1 m. The supernatant consisted predominantly of monomeric nucleosomes, while most dimeric and larger nucleosomes precipitated. A variable percentage of monomers also precipitated, the exact amount depending upon the extent of digestion. The solubility properties can be used for the simple preparation of fractions that are highly enriched for monomers either containing, or deplete in, lysine-rich histone.     

Protease nexin II interactions with coagulation factor XIa are contained within the Kunitz protease inhibitor domain of protease nexin II and the factor XIa catalytic domain

Protease nexin II, a platelet-secreted protein containing a Kunitz-type domain, is a potent inhibitor of factor XIa with an inhibition constant of 250-400 pM. The present study examined the protein interactions responsible for this inhibition. The isolated catalytic domain of factor XIa is inhibited by protease nexin II with an inhibition constant of 437 +/- 62 pM, compared to 229 +/- 40 pM for the intact protein. Factor XIa is inhibited by a recombinant Kunitz domain with an inhibition constant of 344 +/- 37 pM versus 422 +/- 33 pM for the catalytic domain. Kinetic rate constants were determined by progress curve analysis. The association rate constants for inhibition of factor XIa by protease nexin II [(3.35 +/- 0.35) x 10(6) M(-1) s(-1)] and catalytic domain [(2.27 +/- 0. 25) x 10(6) M(-1) s(-1)] are nearly identical. The dissociation rate constants are very similar, (9.17 +/- 0.71) x 10(-4) and (7.97 +/- 1.1) x 10(-4) s(-1), respectively. The rate constants for factor XIa and catalytic domain inhibition by recombinant Kunitz domain are also very similar: association constants of (3.19 +/- 0.29) x 10(6) and (3.25 +/- 0.44) x 10(6) M(-1) s(-1), respectively; dissociation constants of (10.73 +/- 0.84) x 10(-4) and (10.36 +/- 1.3) x 10(-4) s(-1). The inhibition constant (K(i)) values calculated from these kinetic parameters are in close agreement with those measured from equilibrium binding experiments. These results suggest that the major interactions required for factor XIa inhibition by protease nexin II are localized to the catalytic domain of factor XIa and the Kunitz domain of protease nexin II.     

Guinea pig liver transglutaminase: A modified purification procedure affording enzyme with superior activity in greater yield.

Tissue transglutaminase purified from guinea pig livers has a very broad substrate specificity in comparison with other members of the transglutaminase family and therefore is useful for substrate analogue kinetic studies. Modifications made in our laboratory to the standard purification protocol (J. E. Folk and S. I. Chung, 1985, Methods Enzymol. 113, 358-364) have yielded a 28% increase in specific activity and 55% increase in overall yield, while reducing the number of steps to the purification. Herein we report some of the highest yields and specific activities for guinea pig liver transglutaminase found in the literature, as well as the use of lyophilization as a solution to the long-standing problem of enzyme stability during storage.     

Properties of a New Plant Serine Protease Cucumisin

Cucumisin [EC 3.4.21.25] was coupled to cyanogen bromide-activated Sephayose 4B. The specific activity of the immobilized cucumisin was 41% of that of the soluble cucumisin toward casein. The immobilized enzyme was more stable against alkaline inactivation or heat than the soluble enzyme. In using affinity chromatography on a column of the immobilized cucumisin-Sepharose, cucumisin inhibitor was not obtained from potent sources of proteinase inhibitors, such as pig kidney and liver, avian and turtle egg-whites, or soybeans.     

Purification and Some Properties of a Protease from Streptomyces limosus

Streptomyces limosus was selected because it secreted a novel protease that catalyzed the synthetic reaction forming Pro-Pro-Pro from Pro-Pro. The protease was purified to an electrophoretically homogeneous state and an activity of more than about 20,000-fold that of the culture broth. The molecular mass of the enzyme was estimated to be 50 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme was most active in alkaline pH for the synthetic reaction producing Pro-Pro-Pro from Pro-Pro, although for the hydrolytic reaction forming proline it was most active in neutral pH. The enzyme was inhibited by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and diazoacetyl-DL-norleucine methyl ester (DAN). It can be considered that this enzyme belongs to the class of aspartic proteases. The substrate specificity indicates that this enzyme has a strong affinity for proline as a N-terminal amino acid of peptides.     

Ubiquitin with a non-ATP-dependent slow intrinsic proteolytic activity: a mild and rapid purification procedure.

Ubiquitin has been purified to homogeneity, through a dialysis membrane having a NMW cutoff of 12 kDa, by taking advantage of its non-dialysable nature under these conditions. The dialysate was continuously recycled through a CM-52 cation exchange column at pH 4.5. The adsorbed fraction was eluted selectively at pH 7.2. Ubiquitin (25 mg) was obtained from 500 ml of packed RBCs. On SDS PAGE, ubiquitin showed varying mobility depending on the time of boiling in SDS. With 2 min of boiling, the molecular weight seemed to be 10.5 kDa, whereas 10 min of boiling resulted in a molecular weight of 8.5 kDa. Ubiquitin showed a slow intrinsic proteolytic activity against SDS-denatured beta-galactosidase in the absence of ATP. For the first 4 hr, there was no detectable degradation, but degradation was nearly complete after 8 hr. These data are not in agreement with those of Freid et al. [Proc. Natl Acad. Sci, USA, 84 (1987), 3685] who have reported a proteolytic activity comparable to that of other proteolytic enzymes.     

A rapid procedure for purification of EcoRI endonuclease.

A convenient and rapid procedure has been developed to purify restriction endonuclease Eco RI. The method involves sonication of cells at low ionic strength, precipitation of the endonuclease with Polymin P (a polyethyleneimine), elution of the enzyme from the Polymin P precipitate, ammonium sulfate precipitation and chromatography on phosphocellulose. The purified restriction endonuclease is free of exonuclease and other endonucleases.     

Protein inhibitor of acid deoxyribonucleases. Improved purification procedure and properties.

A method is described for the extensive purification of acid deoxyribonuclease (acid DNase) and its specific inhibitor from beef liver, the existence of which had been only supported by indirect evidence. By the use of insolubilized acid deoxyribonuclease, eight other proteins interacting with the enzyme have been detected. One of them (molecular weight, 59,000) was identified as responsible for phosphodiesterase activity which is often a contaminant of DNase preparations. Acid DNase (free of phosphodiesterase) and its inhibitor have been obtained as homogeneous proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of acid DNase and its inhibitor are, respectively, 26,500 and 21,500; those of other proteins range from 17,000 to 112,000. The properties of beef liver acid DNase are similar to those described for the enzymes extracted from other sources. The same alteration of DNase kinetics by this inhibitor, as that previously demonstrated with an impure protein has been confirmed; the sigmoidal shape observed at pH 5 for the plot of initial rate versus substrate concentration progressively disappears with increasing pH. We have also demonstrated that RNA, which inhibits the acid DNase through a competitive binding to the catalytic site, is able, like the substrate, to reverse the binding of inhibitor to the enzyme.