Identification of elastase in human eosinophils: immunolocalization, isolation, and partial characterization.

Although an elastolytic activity in eosinophil-rich cell fractions from mice has been reported, this enzyme has not been purified and characterized as yet in any mammalian species. Eosinophilic elastase was isolated from human eosinophil fragments (cytosomes) obtained from normal and eosinophilic subjects. The enzyme was purified to apparent electrophoretic homogeneity by fast protein liquid chromatography. The enzyme shows the same physical properties of the major elastase isoenzyme of human neutrophils. In addition, like monocyte elastase, it reacts with a monoclonal antibody against human neutrophil elastase. The biochemical similarities observed between the above-mentioned enzymes and the immunolocalization findings strongly support the idea that human eosinophils and neutrophils contain the same enzyme activity. Eosinophils show immunoreactive material in both types of dense cytoplasmic granules. This observation supports the current hypothesis that the different types of eosinophilic granules represent successive morphological stages of maturation.     

Kinesin transports RNA: isolation and characterization of an RNA-transporting granule

RNA transport is an important and fundamental event for local protein synthesis, especially in neurons. RNA is transported as large granules, but little is known about them. Here, we isolated a large RNase-sensitive granule (size: 1000S approximately) as a binding partner of conventional kinesin (KIF5). We identified a total of 42 proteins with mRNAs for CaMKIIalpha and Arc in the granule. Seventeen of the proteins (hnRNP-U, Pur alpha and beta, PSF, DDX1, DDX3, SYNCRIP, TLS, NonO, HSPC117, ALY, CGI-99, staufen, three FMRPs, and EF-1alpha) were extensively investigated, including their classification, binding combinations, and necessity for the "transport" of RNA. These proteins and the mRNAs were colocalized to the kinesin-associated granules in dendrites. The granules moved bidirectionally, and the distally directed movement was enhanced by the overexpression of KIF5 and reduced by its functional blockage. Thus, kinesin transports RNA via this granule in dendrites coordinately with opposite motors, such as dynein.     

Human placental anticoagulant protein: isolation and characterization

An anticoagulant protein was purified from the soluble fraction of human placenta by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose, Sephadex G-75, and Mono S (Pharmacia). The yield of the purified protein was approximately 20 mg from one placenta. The purified protein gave a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 36,500. This protein prolonged the clotting time of normal plasma when clotting was induced either by brain thromboplastin or by kaolin in the presence of cephalin and Ca2+. It also prolonged the factor Xa induced clotting time of platelet-rich plasma but did not affect thrombin-induced conversion of fibrinogen to fibrin. The purified placental protein completely inhibited the prothrombin activation by reconstituted prothrombinase, a complex of factor Xa-factor Va-phospholipid-Ca2+. The placenta inhibitor had no effect on prothrombin activation when phospholipid was omitted from the above reaction. Also, it neither inhibited the amidolytic activity of factor Xa, nor did it bind to factor Xa. The placenta inhibitor, however, did bind specifically to phospholipid vesicles (20% phosphatidylserine and 80% phosphatidylcholine) in the presence of calcium ions. These results indicate that the placental anticoagulant protein (PAP) inhibits coagulation by binding to phospholipid vesicles. The amino acid sequences of three cyanogen bromide fragments of PAP aligned with those of two distinct regions of lipocortin I and II with a high degree of homology, showing that PAP is a member of the lipocortin family.     

Human endometrial stem cells: High-yield isolation and characterization

Background

Menstrual blood is only recently and still poorly studied, but it is an abundant and noninvasive source of highly proliferative mesenchymal stromal cells (MSCs). However, no appropriate isolation method has been reported due to its high viscosity and high content of clots and desquamated epithelium.

Human leukocyte elastase hydrolysis of peptides derived from human elastin exon 24

In normal and pathological tissues, polymorphonuclear leukocyte proteases (elastase, cathepsin G and proteinase 3) may generate soluble peptides through limited proteolysis of elastin, the main component of mature elastic fibres. Elastin-derived peptides display diverse biological activities including cell migration, differentiation, proliferation, chemotaxis, tumor progression and up-regulation of metalloproteinases. To be biologically active, their structures must adopt a beta-turn conformation which accommodates to the cell surface-located elastin binding protein. In this study, we established that human elastin exon 24-derived peptides are hydrolysed by leukocyte elastase, when the active site is fully occupied (from S(5) to S'(3)). As shown by mass spectrometry analyses, a major cleavage site was demonstrated at a Val-Ala bond and a minor one at Gly-Val bond. For longer peptides, the hydrolysed fragments could themselves be re-hydrolysed. If the shortest fragments do not contain the GxxPG sequence known to stimulate cellular effects, some of the intermediates together with hydrolysis fragments generated by other proteases such as proteinase 3, may possess this motif.     

Human leukocyte interferon: Isolation and characterization of several molecular forms

Purification of human leukocyte interferon by high-performance liquid chromatography yielded eight major interferon species. These were characterized by their molecular weight, amino acid composition, and antiviral activity on human and bovine cells. The molecular weights of the purified species ranged from 16,000 to 21,000. The tryptic peptide profiles of all the pure species were compared. Both amino acid compositions and tryptic peptide profiles revealed a structural similarity among the various species.     

Human pancreatic enzymes: purification and characterization of a nonelastolytic enzyme, protease E. resembling elastase.

?An enzyme with proteolytic activity has been isolated from activated extracts of human pancreatic tissue. The purification procedure included salt fractionation followed by ion-exchange chromatography on SE-TSephadex C-25 and on DEAE-Sephadex A-50. The homogeneity of this enzyme, designated protease te, was demonstrated by disc electrophoresis and by sedimentation equilibrium centrifugation stidues. The homogeneous enzyme shows the ability to hydrolyze many of the conventional synthetic substrates used for the identification of elastase activity; however, it demonstrates no significant elastolytic activity. A comparison of human protease E with porcine elastase reveals a high degree of similarity between the two proteases with respect to inhibition by active-site directed peptide chloromethyl ketones, stability, decreased susceptibility to naturally occurring proteinase inhibitors, and specificity for synthetic substrates as well as several other physical properties. The major difference between human protease E and porcine elastase, other than the lack of elastolytic activity by human protease E, seems to be in the ionic character and the amino acid composition of these two proteins. Porcine elastase is a cationic enzyme, while human protease E appears to be anionic in nature. These dissimilarities concerning elastolytic activity and ionic character appear to be directly related.     

The elastase inhibitors of swine serum: isolation and partial characterization of the beta 1-globulin inhibitor and its interaction with elastase

1. The principal elastase inhibitor of swine serum, a beta 1-globulin, has been isolated from serum by ammonium sulfate fractionation, DEAE-cellulose column chromatography and chromatofocusing. 2. The purified beta 1-globulin was homogeneous by immunoelectrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Multiple zones (isoinhibitors) were produced on anionic polyacrylamide gels. The mol. wt for the native, elastase-inactivated inhibitor and the beta 1-globulin-elastase complex were respectively, 65,467 and 60,000 and 79,667. The amino acid residue weight was 63,331. 4. The electrophoretic mobilities of the native inhibitor, elastase-inactivated inhibitor and the inhibitor-elastase complex were respectively, -3.4, -3.8 and -2.2 x 10(-5) cm2/V per sec, the isoelectric points were respectively, 4.78-5.28 (major pIs = 5.15, 5.35), 4.63-5.35 (major pI = 5.13) and 6.02-6.2 (major pI = 6.12). 5. The first order dissociation rate constant for the beta 1-globulin-elastase complex (two-fold molar excess of elastase at 37 degrees C) was 1.9 x 10(-3) per sec with complete dissociation in 40.4 min. The dissociation constant for the complex was 1.47 x 10(-7) M. One mol of elastase was bound per mol of the inhibitor. 6. The beta 1-globulin-elastase complex reacts with antibody to either protein moiety.     

Human C1 inhibitor: improved isolation and preliminary structural characterization

An improved procedure for the isolation of the C1 inhibitor (C1-INH) component of human complement is reported. Following preliminary steps to remove plasminogen, fibrinogen, and aggregated material, three conventional chromatographic steps are used to isolate C1-INH in high (70%) overall yield. An extinction coefficient (E 1%, 1 cm 280nm) of 3.60 has been determined. The isolated protein exhibits a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a mobility corresponding to an apparent molecular weight (Mr) of 105 000. After removal of carbohydrate, the protein shows an increased mobility, corresponding to an apparent Mr of 78 000. A total carbohydrate content of 33% has been calculated, and from this and the size of the deglycosylated polypeptide, a true molecular weight of 116 000 was estimated. Further analysis of the carbohydrate has indicated a galactose:mannose ratio of 2:1 and approximately equimolar amounts of N-acetylglucosamine and N-acetylgalactosamine. This composition is unusual for a plasma protein and suggests that much of the carbohydrate is contained in linkages other than the typical N-glycosidic structures. Values found for the amino acid composition are compared with those reported previously. The amino-terminal sequence (40 residues) of C1-INH is also reported. Asparagine lies at the amino terminus. Neither high-performance liquid chromatography of the released phenylthiohydantoin derivative nor back-hydrolysis of the thiazolinone permitted identification of the residue contained at position 3. The sequence around this position is compatible, however, with an N-glycosidic linkage to residue 3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction between leukocyte elastase and elastin: quantitative and catalytic analyses

Solubilization of elastin by human leukocyte elastase (HLE) cannot be analyzed by conventional kinetic methods because the biologically relevant substrate is insoluble and the concentration of enzyme-substrate complex has no physical meaning. We now report quantitative measurements of the binding and catalytic interaction between HLE and elastin permitted by analogy to receptor-ligand systems. Our results indicated that a limited and relatively constant number of enzyme binding sites were available on elastin, and that new sites became accessible as catalysis proceeded. The activation energies and solvent deuterium isotope effects were similar for catalysis of elastin and a soluble peptide substrate by HLE, yet the turnover number for HLE digestion of elastin was 200-2000-fold lower than that of HLE acting on soluble peptide substrates. Analysis of the binding of HLE to elastin at 0 degrees C, in the absence of significant catalytic activity, demonstrated two classes of binding sites (Kd=9.3x10(-9) M and 2.5x10(-7) M). The higher affinity sites accounted for only 6% of the total HLE binding capacity, but essentially all of the catalytic activity, and dissociation of HLE from these sites was minimal. Our studies suggest that interaction of HLE with elastin in vivo may be very persistent and permit progressive solubilization of this structurally important extracellular matrix component.     

Human pituitary thyrotropin: isolation and chemical characterization of its subunits

The subunits of human pituitary thyrotropin have been separated and purified by countercurrent distribution and exclusion chromatography. The NH₂-terminal sequence of the β subunit is identical to that of the β subunit of bovine thyrotropin. However, amino acid composition and peptide map of tryptic and chymotryptic digests as well as compositions of tryptic and chymotryptic peptides suggest that the amino acid sequence of the α subunit is identical to that of the α subunit of human interstitial cell stimulating hormone.     

Human lysosomal elastase. Catalytic and immunological properties.

1. The elastase of human spleen was shown to exhibit endopeptidase activity against azo-casein and elastin. 2. Activity against several synthetic substrates was detected, and benzyloxycarbonyl-L-alanine 2-naphthyl ester was found to be a good substrate for routine use. 3. The enzyme showed a broad pH optimum in the range of 8.2-9.2 against azo-casein and the synthetic substrate. 4. The effect of inhibitors on the spleen elastase showed it to be a serine proteinase with a specificity similar to that of porcine pancreatic elastase. 5. Specific antisera were raised against the enzyme, and it was shown to be immunologically identical with the lysosomal elastase of human neutrophil leucocytes.     

Interaction between human leukocyte elastase and chondroitin sulfate

Chondroitin sulfate (Structum) interacts with human leukocyte elastase, a potent mediator of articular cartilage degradation, producing a partial inhibition of the enzyme activity (60% at saturation). Kinetically, the inhibition mechanism can be classified as simple intersecting, hyperbolic noncompetitive and is almost identical to that found earlier for similar compounds. The best inhibitory activity of chondroitin sulfate was found in fractions having at the same time a high proportion of chondroitin-6-sulfate relative to the corresponding 4-isomer and a high molecular mass. Thus, a fraction with high Mr and containing 92% of isomer 6 inhibited leukocyte elastase with Ki = 1.8 micrograms/ml, whereas a fraction with low Mr and almost equal composition of the 4- and 6-isomer had Ki = 140 micrograms/ml. Ki for unfractionated chondroitin sulfate was 3.4 micrograms/ml. It is suggested, that the modulation of the extracellular activity of cartilage-degrading enzymes by cartilage-derived factors may explain, at least in part, the beneficial effects of some therapeutically used chondroprotective agents.     

Mouse liver cytoplasmic malate dehydrogenase: rapid isolation, characterization and immunochemical comparison with hepatoma enzyme.

Cytoplasmic NAD-dependent malate dehydrogenase is decreased in activity in three transplantable mouse hepatomas compared to the activity of this enzyme in liver tissue. This enzyme is composed of several molecular forms of similar size which differ slightly in charge; the total activity and the discernible number of forms of the enzyme are decreased in both hepatoma and fetal liver. Mixing experiments suggest the absence of a significant quantity of unbound inhibitor of enzyme activity in the tumor or an activator in the liver. The liver cytoplasmic enzyme was purified to homogeneity by a relatively rapid method using Blue Sepharose affinity chromatography, which results in a good yield and high specific activity of the enzyme. Cytoplasmic and mitochondrial enzymes bind and elute differentially from this affinity resin. Molecular weight, kinetic constants and amino acid composition of the cytoplasmic enzyme were determined. Monospecific antiserum to the cytoplasmic enzyme has been produced in a goat and used to demonstrate a lack of immunological cross-reactivity between the mitochondrial and cytoplasmic enzyme. The tumor and liver cytoplasmic enzymes possess similar, if not identical, immunological determinants. Immunotitration experiments have been used to demonstrate that liver and hepatoma enzyme are identical in specific activity. Thus, the reduction in level of cytoplasmic enzyme in hepatoma is due to a decrease in the number of molecules per tissue mass.     

Human epithelial cell intermediate filaments: isolation, purification, and characterization

Intermediate filaments (IF) isolated from human epithelial cells (HeLa) can be disassembled in 8 M urea and reassembled in phosphate-buffered solutions containing greater than 0.1 mg/ml IF protein. Eight proteins were associated with HeLa IF after several disassembly-reassembly cycles as determined by sodium dodecyl sulfate gel electrophoresis (SDS PAGE). A rabbit antiserum directed against HeLa IF contained antibodies to most of these proteins. The immunofluorescence pattern that was seen in HeLa cells with this antiserum is complex. It consisted of a juxtanuclear accumulation of IF protein and a weblike array of cytoplasmic fibers extending to the cell border. Following preadsorption with individual HeLa IF proteins, the immunofluorescence pattern in HeLa cells was altered to suggest the presence of at least two distinct IF networks. The amino acid composition and alpha-helix content (approximately 38%) of HeLa IF proteins was similar to the values obtained for other IF proteins. One-dimensional peptide maps show extensive homology between the major HeLa IF protein of 55,000-mol- wt and a similar 55,000-mol-wt protein obtained from hamster fibroblasts (BHK-21). HeLa 55,000-mol-wt homopolymer IF assembled under conditions similar to those required for BHK-21 55,000-mol-wt homopolymers. Several other proteins present in HeLa IF preparations may be keratin-like structural proteins. The results obtained in these studies indicate that the major HeLa IF protein is the same major IF structural protein found in fibroblasts. Ultrastructural studies of HeLa cells revealed two distinct IF organizational stages including bundles and loose arrays. In addition, in vitro reconstituted HeLa IF also exhibited these two organizational states.