Fibrino(geno)lytic Properties of Purified Hementerin, a Metalloproteinase from the Leech Haementeria depressa

1998 ◽  
Vol 80 (07) ◽  
pp. 155-160 ◽  
Author(s):  
Ana Marisa Chudzinski-Tavassi ◽  
Eva Maria Kelen ◽  
Ana Paula de Paula Rosa ◽  
Stephane Loyau ◽  
Claudio Sampaio ◽  
...  
Keyword(s):  
Gel Filtration ◽  
Exchange Chromatography ◽  
Single Chain ◽  
Terminal Sequence ◽  
Calcium Dependent ◽  
Amino Terminal ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Amino Terminal Sequence

SummaryThe fibrino(geno)lytic protein designated hementerin contained in crude extracts of the salivary complex of Haementeria depressa leeches was purified to apparent homogeneity by gel filtration, ion exchange chromatography and preparative SDS-PAGE. It is a single-chain 80 kDa, PhMeSO2F-resistant, calcium-dependent, metalloproteinase, which specifically degrades fibrin(ogen) through a plasminogen-independent pathway. The amino terminal sequence of 8 residues shows 80% similarity with hementin, another fibrino(geno)lytic protein purified from Haementeria ghilianii leeches. However, their activities differ somewhat in terms of kinetics and with regard to the structure of the fibrin(ogen) fragments they may produce. Cleavage by hementerin of fibrinogen Aα, γ and Bβ chains, in that order, produces 270 kDa to 67 kDa fragments which differ from those produced by plasmin. Hementerin was also able to degrade cross-linked fibrin although at a lower rate as compared to fibrinogen. In conclusion, hementerin is a plasminogen-independent fibrino(geno)lytic metalloproteinase that degrades fibrinogen faster than fibrin, prevents blood coagulation and destroys fibrin clots in vitro.

Purification and characterization of prostate-specific antigen (PSA) complexed to alpha 1-antichymotrypsin: potential reference material for international standardization of PSA immunoassays

1995 ◽  
Vol 41 (9) ◽  
pp. 1273-1282 ◽  
Author(s):  
Z Chen ◽  
A Prestigiacomo ◽  
T A Stamey
Keyword(s):  
Molecular Mass ◽  
Prostate Specific Antigen ◽  
Specific Antigen ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Molar Ratio ◽  
Free Psa ◽  
Apparent Homogeneity ◽  
International Standardization

Abstract We describe for the first time a protocol to purify to apparent homogeneity an in vitro-prepared complex of prostate-specific antigen (PSA) and alpha 1-antichymotrypsin (ACT) by using a combination of gel filtration and ion-exchange chromatography. The purity of the PSA-ACT complex was confirmed by gel electrophoresis and Western blot. The PSA-ACT complex was stable in the pH range 6.0 to 7.8; it was also stable in various matrices, temperatures, and high concentrations of salt. Purification of the PSA-ACT complex was highly reproducible. An absorptivity of 0.99 L x g-1 x cm-1 at 280 nm was assigned to the PSA-ACT complex, based on amino acid analysis. Because PSA and ACT bind in a 1:1 molar ratio, we determined the molecular mass of the PSA-ACT complex as the mass encoded by the cDNA of ACT (plus 26% carbohydrate) plus the molecular mass of PSA (28,430 Da), which totals 89,280 Da. Using this material, we made two common calibrators, one of 100% PSA-ACT complex and one of 90% PSA-ACT complex plus 10% free PSA by volume (90:10 calibrator). Substitution of these calibrators for the manufacturers' calibrators in nine commercial immunoassays substantially reduced differences between immunoassays, especially for serum PSA values between 4 and 10 micrograms/L. The 90:10 calibrator is recommended as a universal calibrator for international standardization of PSA immunoassays.

Partial primary sequence of a human endometrial epithelial progesterone dependent protein secreted in vitro

1991 ◽  
Vol 124 (3) ◽  
pp. 338-345 ◽  
Author(s):  
Helen L. Dichek ◽  
David T. MacLaughlin ◽  
F. Richard Bringhurst ◽  
Henry T. Keutmann ◽  
George S. Richardson
Keyword(s):  
Luteal Phase ◽  
High Performance ◽  
Compositional Analysis ◽  
Single Individual ◽  
Terminal Sequence ◽  
Step Method ◽  
Two Dimensional Gel Electrophoresis ◽  
Amino Terminal ◽  
Amino Terminal Sequence

Abstract. We have recently identified a uterine progesterone-dependent 25 kD protein (protein #27) secreted by endometrial epithelial cells but not stroma. Protein #27 was originally isolated from human luteal phase uterine fluids and partially purified by two-dimensional gel-electrophoresis and electroelution. It could not be detected in serum. We here report the further purification and amino terminal sequence of this protein, obtained from in vitro luteal phase endometrial tissue incubation media. Protein #27 was purified by a one-step method using reverse-phase high performance liquid chromatography. Amino acid compositional analysis confirms a high content of nonpolar and hydrophobic residues (40 mol%) and reveals a preponderance of acidic amino acids, consistent with its isoelectric point (5.9-6.3 pH). The amino terminal sequence obtained from a single individual reveals 85% homology with that of pregnancy-associated α2-globulin from pooled cytosolic fractions from pregnancy endometria. We propose that protein #27, secreted by the endometrial epithelium, is an excellent candidate for a marker to be used to study pathological processes of the endometrium such as endometriosis and invasive carcinoma.

Purification and characterization of different molecular forms of prostate-specific antigen in human seminal fluid

1995 ◽  
Vol 41 (11) ◽  
pp. 1567-1573 ◽  
Author(s):  
W M Zhang ◽  
J Leinonen ◽  
N Kalkkinen ◽  
B Dowell ◽  
U H Stenman
Keyword(s):  
Enzymatic Activity ◽  
Prostate Specific Antigen ◽  
Seminal Fluid ◽  
Specific Antigen ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Terminal Sequence ◽  
Amino Terminal ◽  
Intact Psa ◽  
Amino Terminal Sequence

Abstract We have developed a new procedure for purifying prostate-specific antigen (PSA) from human seminal fluid. The method is based on ammonium sulfate precipitation, hydrophobic interaction chromatography, gel filtration, and anion-exchange chromatography. It can be completed within 2 days with a recovery of intact PSA of 30%. By anion-exchange chromatography, five isoforms of PSA (A, B, C, D, and E) can be separated. The major form (PSA-B) consists of the intact enzyme, as shown by the occurrence of only one band of 33 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or nonreducing conditions, and by amino acid sequencing, which reveals only one amino-terminal sequence corresponding to the reported amino-terminal sequence of intact PSA. The specific absorbance of 1 g/L PSA-B at 280 nm was 1.61, and 80% of the PSA-B formed a complex with alpha 1-antichymotrypsin, indicating that it is enzymatically active. Three cleaved forms of PSA with different nicking sites and low enzymatic activity were separated from intact PSA by ion-exchange chromatography. In addition, we isolated a glycosylation variant, PSA-A, which showed a higher isoelectric point (pI = 7.2) than PSA-B (pI = 6.9) but similar enzymatic activity; this form accounts for 5-10% of total PSA. After treatment with sialidase, PSA-A and B had the same isoelectric point value (pI = 7.7).

Purification and characterization of a β-glucosidase from solid-state cultures ofHumicola griseavar.thermoidea

1996 ◽  
Vol 42 (1) ◽  
pp. 1-5 ◽  
Author(s):  
Edivaldo Ximenes Ferreira Filho
Keyword(s):  
Solid State ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Performance Liquid Chromatographic ◽  
Exchange Chromatography ◽  
Glucosidase Activity ◽  
Apparent Homogeneity ◽  
Sds Page

The thermophilic fungus Humicola grisea var. thermoidea produced β-glucosidase activity when grown in a solid-state culture on wheat bran as carbon source. A β-glucosidase was purified to apparent homogeneity by ultrafiltration, gel filtration chromatography on Sephacryl S-100, and ion-exchange chromatography on S-Sepharose, as judged by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) on a 12.5% (w/v) slab gel. The enzyme had a molecular mass of 82 and 156 kDa, as estimated by SDS–PAGE and gel filtration on a high performance liquid chromatographic column, respectively, suggesting that the native enzyme may consist of two identical subunits. The purified enzyme was thermostable at 60 °C for 1 h with a half-life of 15 min at 65 °C, and displayed optimum activity at 60 °C and a pH range of 4.0–4.5. The Kmand Vmaxvalues for p-nitrophenyl β-D-glucopyranoside were determined to be 0.316 mM and 0.459 IU∙mL−1, respectively. D-Glucose, D-gluconic acid lactone, Hg2+, Cu2+, and Mn2+inhibited β-glucosidase activity. The enzyme activity was competitively inhibited by D-glucose (Ki = 0.6 mM). The purified enzyme was very active against cellobiose and p-nitrophenyl β-D-glucopyranoside.Key words: Humicola, β-glucosidase, purification, characterization.

scholarly journals A variant of prealbumin from amyloid fibrils in familial polyneuropathy of Jewish origin.

1981 ◽  
Vol 154 (3) ◽  
pp. 989-993 ◽  
Author(s):  
M Pras ◽  
E C Franklin ◽  
F Prelli ◽  
B Frangione
Keyword(s):  
Amino Acid ◽  
Amyloid Fibrils ◽  
Gel Filtration ◽  
Familial Amyloidotic Polyneuropathy ◽  
Antigenic Determinants ◽  
Terminal Sequence ◽  
Jewish Origin ◽  
Amino Terminal ◽  
Amino Terminal Sequence ◽  
Human Prealbumin

Amyloid fibrils were isolated from spleen and thyroid obtained at autopsy from one patient (S.K.O.) of Jewish origin with familial amyloidotic polyneuropathy. Gel filtration on Sephadex G100 after solubilization in 5 M guanidine HCl yielded three major components with 14,000, 9,000, and 5,000 mol wt, respectively. The two larger components shared antigenic determinants with human prealbumin. Amino acid analysis and amino terminal sequence studies revealed the 14,000-mol wt protein to be an intact prealbumin subunit. The 9,000-mol wt fragment obtained in highest yield encompassed the region from position 49-127 and the 5,000 mol wt fraction encompassed the amino terminal of prealbumin (position 1-48). An amino acid substitution (Gly/Thr) was detected at position 49, where enzymatic cleavage occurred. Thus, several prealbumin-derived fragments, predominantly the carboxyl end, constitute the amyloid fibrils in a heredofamilial amyloidosis syndrome of dominant inheritance.

scholarly journals Purification and characterization of the major iron-regulated protein expressed by pathogenic Neisseriae.

1987 ◽  
Vol 165 (4) ◽  
pp. 1041-1057 ◽  
Author(s):  
T A Mietzner ◽  
G Bolan ◽  
G K Schoolnik ◽  
S A Morse
Keyword(s):  
Gel Filtration ◽  
Exchange Chromatography ◽  
Hexadecyltrimethylammonium Bromide ◽  
Direct Role ◽  
Biologically Relevant ◽  
Amino Terminal ◽  
Porin Protein ◽  
Iron Assimilation ◽  
Sds Page

This report describes a method to purify the major iron-regulated protein (MIRP) expressed by N. gonorrhoeae and N. meningitidis. This purification procedure involves maximal expression of the MIRP by growing the organisms on iron-limited media; cellular disruption by sonication followed by centrifugal fractionation; selective solubilization of the MIRP with the cationic detergent hexadecyltrimethylammonium bromide; cation-exchange chromatography in the presence of this detergent; and gel filtration chromatography. The MIRP purified by this technique migrates as a single band when analyzed by SDS-PAGE. The purified MIRP displayed an unusually basic isoelectric point, this value being greater than 9.35. Further biochemical analysis revealed the highly conserved nature of this protein isolated from the two pathogenic species of the genus Neisseria. For example, the amino acid composition of the meningococcal and gonococcal MIRPs were nearly identical and amino terminal sequence analysis showed that both shared the identical primary sequence through residue 48. Surprisingly, the first five NH2-terminal residues of the MIRPs exhibited homology with the first five residues of the gonococcal porin, protein I. Purified preparations of the MIRP exhibited a characteristic pink color reminiscent of the basic iron-binding protein lactoferrin. This observation coupled with the property of iron-regulation prompted us to analyze purified MIRP for iron-content. Approximately 0.5 mol iron per 1 mol of MIRP was detected. This study is the first to show that iron is associated with the MIRP, a property that may implicate this protein as playing a direct role in neisserial iron assimilation. While the precise function of the MIRP is not known, the availability of this protein in pure and biologically relevant quantities will allow further studies to elucidate its pathobiologic function.

scholarly journals Actin-Binding Proteins from Burkholderia mallei and Burkholderia thailandensis Can Functionally Compensate for the Actin-Based Motility Defect of a Burkholderia pseudomallei bimA Mutant

2005 ◽  
Vol 187 (22) ◽  
pp. 7857-7862 ◽  
Author(s):  
Joanne M. Stevens ◽  
Ricky L. Ulrich ◽  
Lowrie A. Taylor ◽  
Michael W. Wood ◽  
David DeShazer ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Binding Proteins ◽  
Actin Binding ◽  
Burkholderia Mallei ◽  
Terminal Sequence ◽  
Actin Binding Proteins ◽  
Burkholderia Thailandensis ◽  
Amino Terminal ◽  
Amino Terminal Sequence

ABSTRACT Recently we identified a bacterial factor (BimA) required for actin-based motility of Burkholderia pseudomallei. Here we report that Burkholderia mallei and Burkholderia thailandensis are capable of actin-based motility in J774.2 cells and that BimA homologs of these bacteria can restore the actin-based motility defect of a B. pseudomallei bimA mutant. While the BimA homologs differ in their amino-terminal sequence, they interact directly with actin in vitro and vary in their ability to bind Arp3.

Preparation and characterization of the N and C monoferric lobes of buffalo lactoferrin produced by proteolysis using proteinase K

1999 ◽  
Vol 66 (1) ◽  
pp. 81-90 ◽  
Author(s):  
SUJATA SHARMA ◽  
TEJ P. SINGH ◽  
KRISHAN L. BHATIA
Keyword(s):  
Molecular Mass ◽  
Limited Proteolysis ◽  
Hydrolysis Product ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Proteinase K ◽  
Single Chain ◽  
Helical Structures ◽  
Sds Page ◽  
Clear Band

The two glycosylated N- and C-terminal lobes of buffalo lactoferrin have been produced by limited proteolysis using proteinase K. Lactoferrin is a single chain glycoprotein of molecular mass 80 kDa with two iron-binding sites and two structural lobes connected by a short peptide. Purified samples of lactoferrin, isolated from buffalo colostrum, were subjected to hydrolysis using trypsin, chymotrypsin, pepsin, subtilisin and proteinase K. The first three proteinases produced two major fragments of approximately 35 and 23 kDa together with small molecular mass peptides. Trypsin and chymotrypsin partly digested lactoferrin, while pepsin converted all the intact lactoferrin into fragments. Subtilisin hydrolysis produced fragments of 40 and 26 kDa together with low molecular mass peptides. However, SDS-PAGE of the proteinase K hydrolysis product gave a clear band at 40 kDa together with a band indicating a substantial quantity of low molecular mass peptides (<14·4 kDa). Upon ion-exchange chromatography this product gave two major fractions, which were further purified by gel filtration and identified as the C and N lobes from their N-terminal sequences. Thus, the 40 kDa band in SDS-PAGE of the proteinase K hydrolysis product contained two fragments of equal molecular mass. On further hydrolysis with proteinase K, the N lobe was completely hydrolysed into low molecular mass peptides, while only a small fraction of the C lobe was converted into small products. This suggested that an inhibitory fragment was present in the C lobe that was released on hydrolysis to small fragments and prevented complete digestion of the C lobe by high-affinity binding to the active site of proteinase K. This fragment was isolated from the lactoferrin–proteinase K complex and its sequence determined to be Val–Ala–Gln–Gly–Gly–Ala–Ala–Gly–Leu–Ala. Circular dichroism studies indicated a high α-helical content in the native lactoferrin while comparatively lower helical structures were present in the N and C lobes. In addition, the iron saturations of the N and C lobes appeared to be lower than that of the native protein.

scholarly journals Calpactins: two distinct Ca++-regulated phospholipid- and actin-binding proteins isolated from lung and placenta.

1987 ◽  
Vol 104 (3) ◽  
pp. 503-511 ◽  
Author(s):  
J R Glenney ◽  
B Tack ◽  
M A Powell
Keyword(s):  
Binding Sites ◽  
Human Fibroblasts ◽  
Actin Binding ◽  
Terminal Sequence ◽  
Calcium Dependent ◽  
Amino Terminal ◽  
Lower Molecular Mass ◽  
Double Label ◽  
Ca Ions ◽  
Amino Terminal Sequence

Three forms of calpactin, the 36,000 Mr Ca++-binding cytoskeletal protein, were isolated in large amounts from bovine lung and human placenta using cycles of calcium-dependent precipitation followed by solubilization with EGTA-containing buffers. Calpactin-I as a tetramer of heavy (36 kD) and light (11 kD) chains was the predominant form of calpactin isolated, however milligram amounts of the calpactin-I heavy chain monomer and calpactin-II, a related but distinct molecule, were also isolated by this method. Calpactin-II was characterized in some detail and found to bind two Ca++ ions with Kd's of 10 microM in the presence of phosphatidylserine. Both calpactin-I and -II were found to aggregate liposomes at micromolar Ca++ concentrations, suggesting that at least two phospholipid-binding sites are present on these molecules. Both calpactin monomers bind to and bundle actin filament at high (1 mM) but not low (less than 1 microM) Ca++ concentrations. Amino-terminal sequence analysis of a lower molecular mass variant of calpactin-II revealed that this protein was the previously identified human "lipocortin" molecule. Antibodies were elicited to calpactin-I and -II and the cell and subcellular distribution of each was compared. Calpactin-II was only present at high levels in tissues (lung, placenta) which contained high levels of calpactin-I. Other tissues (intestine) contained high calpactin-I and undetectable levels of calpactin-II. Double-label immunofluorescence microscopy on human fibroblasts revealed that, like calpactin-I, calpactin-II is present in a submembraneous reticular network, although the distribution of the two calpactins is not identical.

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