Isolation and purification of a high molecular weight substance from human urine which inhibits calcium oxalate crystal growth

A high molecular weight inhibitor of calcium oxalate crystal growth in human urine was investigated. Three inhibitors were isolated by DEAE-Sephacel ion-exchange chromatography and, of these, the substance we named "Peak 3 protein" seemed to be the main inhibitor in human urine. Peak 3 protein and several was purified by fast protein liquid chromatography and polyacrylamide gel electrophoresis. This substance, with a molecular weight of 30 kDa, did not contain uronic acid and its inhibitory activity decreased after digestion with proteinase. The difference between Peak 3 protein and several inhibitors previously reported was investigated but no clear difference could be found. The fact that it was the protein structure which was responsible for the inhibitory activity and the fact that Peak 3 protein probably possessed many side-chains which did not contribute to the inhibitory activity influenced the outcome of the investigation.     

A probarley lectin processing enzyme purified from Arabidopsis thaliana seeds

An aspartic proteinase was purified from the seeds of Arabidopsis thaliana (ecotype RLD) using affinity chromatography on pepstatin-agarose and ion exchange chromatography. The purified enzyme is optimally active at pH 3.5 and completely inhibited by pepstatin A. The purified Arabidopsis aspartic proteinase contains four subunits (apparent molecular weights 31, 28.5, 15 and 6 kDa), two of which are probably linked by disulfide bridges. These properties are similar to the aspartic proteinase previously isolated from barley seeds. The amino acid sequence of the peptide subunits corresponds exactly with the sequence of the previously isolated cDNA for the Arabidopsis aspartic proteinase. The Arabidopsis enzyme processed probarley lectin in vitro at the carboxy-terminus between phenylalanine and alanine, the same place where the barley enzyme cleaves the lectin in vitro. The aspartic proteinase appears to be the major enzyme processing the lectin in seeds as pepstatin A inhibited this activity in a crude seed extract.     

Purification and cDNA cloning of a four-domain Kazal proteinase inhibitor from crayfish blood cells

A cDNA with an open reading frame of 684 base pairs was isolated from a library from blood cells of the crayfish Pacifastacus leniusculus. It codes for a signal sequence and a mature protein of 209 amino acids with a predicted molecular mass of 22.7 kDa. The amino acid sequence consists of four repeated stretches (45-73% identical to each other), indicating that the protein has four domains. The domains have significant sequence similarity to serine proteinase inhibitors of the Kazal family. The three first domains have a leucine residue in the putative reactive site, suggesting that the protein is a chymotrypsin inhibitor. A monomeric 23-kDa proteinase inhibitor, which by amino terminal sequencing of the mature protein was confirmed to be the cloned Kazal inhibitor, was purified from crayfish blood cells. It inhibited chymotrypsin or subtilisin, but not trypsin, elastase or thrombin. The inhibitor seemed to form a 1:1 complex with chymotrypsin or subtilisin. This protein seems to be the first described Kazal inhibitor from blood cells of any animal and the first one with four domains.     

Purification and identification of a novel and four known serine proteinase inhibitors secreted by human glioblastoma cells

Our previous studies have shown that some human cancer cell lines produce pancreatic trypsinogen, plasminogen, and tissue-type kallikrein. To understand the regulatory mechanism of these proteinases, serine proteinase inhibitors secreted by human glioblastoma cell line T98G were analyzed by gelatin reverse zymography with trypsin. The serum-free conditioned medium of T98G cells showed more than ten trypsin inhibitor bands ranging from 16 to 150 kDa in the reverse zymography. Major trypsin inhibitors were purified by trypsin-affinity chromatography. Analysis of their N-terminal amino acid sequences demonstrated that the purified inhibitors were identical to the secreted forms of amyloid protein precursors (APPs), tissue factor pathway inhibitor (TFPI), placental protein 5 (PP5)/TFPI-2, and secretory leukocyte proteinase inhibitor (SLPI). In addition, a novel 25-kDa trypsin-binding protein, tentatively named p25TI, was identified. p25TI showed weak inhibitory activity against trypsin in reverse zymography as compared with the other inhibitors. The secretion of multiple forms of serine proteinase inhibitors by human cancer cells raises the possibility that they might be involved in the abnormal growth of cancer cells.     

NASBA technology: isothermal RNA amplification in qualitative and quantitative diagnostics

The phosphorylation state of neurofilaments plays an important role in the control of cytoskeletal integrity, axonal transport, and axon diameter. Immunocytochemical analyses of spinal cord revealed axonal localization of all protein phosphatase subunits. To determine whether protein phosphatases associate with axonal neurofilaments, neurofilament proteins were isolated from bovine spinal cord white matter by gel filtration. approximately 15% of the total phosphorylase a phosphatase activity was present in the neurofilament fraction. The catalytic subunits of PP1 and PP2A, as well as the A and B alpha regulatory subunits of PP2A, were detected in the neurofilament fraction by immunoblotting, whereas PP2B and PP2C were found exclusively in the low molecular weight soluble fractions. PP1 and PP2A subunits could be partially dissociated from neurofilaments by high salt but not by phosphatase inhibitors, indicating that the interaction does not involve the catalytic site. In both neurofilament and soluble fractions, 75% of the phosphatase activity towards exogenous phosphorylase a could be attributed to PP2A, and the remainder to PP1 as shown with specific inhibitors. Neurofilament proteins were phosphorylated in vitro by associated protein kinases which appeared to include protein kinase A, calcium/calmodulin-dependent protein kinase, and heparin-sensitive and -insensitive cofactor-independent kinases. Dephosphorylation of phosphorylated neurofilament subunits was mainly (60%) catalyzed by associated PP2A, with PP1 contributing minor activity (10-20%). These studies suggest that neurofilament-associated PP1 and PP2A play an important role in the regulation of neurofilament phosphorylation.     

Brain aging in normotensive and hypertensive strains of rats. III. A quantitative study of cerebrovasculature

The mechanism of degradation of fructose-1,6-bisphosphate aldolase from rabbit muscle by the lysosomal proteinase cathepsin B was determined. Treatment of aldolase with cathepsin B destroys up to 90% of activity with fructose 1,6-bisphosphate as substrate, but activity with fructose 1-phosphate is slightly increased. Cathepsin L, another lysosomal thiol proteinase, and papain are also potent inactivators of aldolase, whereas inactivation is not caused by cathepsins D or H even at high concentrations, or by cathepsin B inhibited by leupeptin or iodoacetate. The cathepsin-B-treated aldolase shows no detectable change in subunit molecular weight, oligomer molecular weight or subunit interactions. Cathepsin B cleaves dipeptides from the C-terminus of th aldolase subunits. Four dipeptides are released sequentially: Ala-Tyr, Asn-His, Ile-Ser and Leu-Phe, and a maximum of five additional dipeptides may be released. There are indications that this peptidyldipeptidase activity of cathepsin B may be an important aspect of its action on protein substrates generally.     

Partial characterization of two serologically related DNA-binding proteins specified by the cottontail rabbit herpesvirus CTHV

We have previously described two serologically related DNA-binding phosphoproteins of different apparent molecular mass (75 kDa and 35 kDa) produced in cottontail rabbit herpesvirus (CTHV)-infected cells. The 75 kDa protein appeared before the 35 kDa protein in the infectious cycle. Here, we extend the characterization of these proteins. Protease V8 fingerprints of methionine-labelled 35 kDa protein showed four major peptide products, three of which co-migrated with major peptides from digests of the 75 kDa protein. The fourth peptide, with an estimated mass of 10 kDa, reacted with an antiserum recognizing both proteins. In vitro translation of total or poly A-containing RNA isolated from infected cells at 24 h to 72 h post-infection produced only the 75 kDa protein as measured by immunoprecipitation with anti-75/35 kDa serum, suggesting that the 35 kDa protein is derived from the 75 kDa protein by proteolytic cleavage. Virus-specific RNA obtained by prehybridization to CTHV DNA also produced the 75 kDA protein, confirming its viral origin. The putative gene for the 75 kDa protein was localized to a region on the CTHV DNA restricted by PvuII.     

Mitochondrial NADH:ubiquinone oxidoreductase (complex I): proximity of the subunits of the flavoprotein and the iron-sulfur protein subcomplexes

The proximities of the three subunits (51, 24, and 9 kDa) of the flavoprotein subcomplex (FP) and five subunits (75, 49, 30, 18, and 13) of the iron-sulfur protein subcomplex (IP) of the bovine NADH: ubiquinone oxidoreductase (complex I) were investigated by cross-linking studies. The cross-linking reagents used were disuccinimidyl tartrate and ethylene glycol bis(succinimidyl succinate). The cross-linked products were identified by sodium dodecyl sulfate gel electrophoresis and immunoblotting with antibodies specific for each subunit. Results showed that the three FP subunits are juxtaposed to one another, and only the 51 kDa subunit of FP is in close proximity to only the 75-kDa subunit of IP. The 75-kDa subunit cross-linked to the 30- and the 13-kDa subunits, the 49-kDa subunit cross-linked to the 30-, 18-, and 13-kDa subunits, and the 30-kDa subunit cross-linked to the 18- and the 13-kDa subunits. No cross-linked products of 75+49-, 75+18-, or 18+13-kDa subunits were detected. These results are consistent with the occurrence of potential electron carriers in FP and IP subunits. These electron carriers are FMN and one iron-sulfur cluster in the 51-kDa subunit, one iron-sulfur cluster in the 24-kDa subunit, and apparently two iron-sulfur clusters in the 75-kDa subunit.     

Purification and properties of the F1F0 ATPase of Ilyobacter tartaricus, a sodium ion pump

The ATPase of Ilyobacter tartaricus was solubilized from the bacterial membranes and purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed the usual subunit pattern of a bacterial F1F0 ATPase. The polypeptides with apparent molecular masses of 56, 52, 35, 16.5, and 6.5 kDa were identified as the alpha, beta, gamma, epsilon, and c subunits, respectively, by N-terminal protein sequencing and comparison with the sequences of the corresponding subunits from the Na(+)-translocating ATPase of Propionigenium modestum. Two overlapping sequences were obtained for the polypeptides moving with an apparent molecular mass of 22 kDa (tentatively assigned as b and delta subunits). No sequence could be determined for the putative a subunit (apparent molecular mass, 25 kDa). The c subunits formed a strong aggregate with the apparent molecular mass of 50 kDa which required treatment with trichloroacetic acid for dissociation. The ATPase was inhibited by dicyclohexyl carbodiimide, and Na+ ions protected the enzyme from this inhibition. The ATPase was specifically activated by Na+ or Li+ ions, markedly at high pH. After reconstitution into proteoliposomes, the enzyme catalyzed the ATP-dependent transport of Na+, Li+, or Hi+. Proton transport was specifically inhibited by Na+ or Li+ ions, indicating a competition between these alkali ions and protons for binding and translocation across the membrane. These experiments characterize the I. tartaricus ATPase as a new member of the family of FS-ATPases, which use Na+ as the physiological coupling ion for ATP synthesis.     

Characterization, purification and cDNA cloning of a rat perchloric-acid-soluble 23-kDa protein present only in liver and kidney

A novel protein was extracted with 5% perchloric acid from rat liver and kidney. It is absent from other rat organs. Its apparent molecular mass is 23 kDa as determined by HPLC gel filtration. A single band, corresponding to 10 kDa, was observed after SDS/PAGE, suggesting that the protein consists of two subunits with similar molecular masses. This protein can neither be phosphorylated by ATP, nor acetylated. The sequence of the cDNA encoding this protein was determined. Southern-blot analysis showed that the corresponding gene spanned at least 10 kb and contained at least five introns. Zoo-blot analysis at medium stringency strongly suggests that the gene has been conserved during evolution. The amino-acid sequence of this protein with a highly conserved region is similar to that of a heat-shock protein.     

Purification and characterization of myonase from X-chromosome linked muscular dystrophic mouse skeletal muscle

A chymotrypsin-like proteinase, designated myonase, was successfully purified to homogeneity from X-chromosome linked muscular dystrophic mouse skeletal muscle by affinity chromatography on agarose conjugated with lima bean trypsin inhibitor as ligand. The molecular mass of the purified myonase was determined to be 26 kDa by SDS-PAGE and to be 25,187 Da by mass spectrometry. The native enzyme is a single chain molecule and a monomeric protein without sugar side-chains. The nucleotide sequence of myonase mRNA is similar to mouse mast cell proteinase 4 (MMCP-4) cDNA. This is the first report of a native enzyme whose amino acid sequence closely corresponds to MMCP-4 cDNA. Myonase has chymotrypsin-like activities and hydrolyzes the amide bonds of synthetic substrates having Tyr and Phe residues at the P1 position. Myonase is most active at pH 9 and at high concentration of salts. Myonase preferentially hydrolyzes the Tyr4-Ile5 bond of angiotensin I and the Phe20-Ala21 bond of amyloid beta-protein, and it is less active towards the Phe8-His9 bond of angiotensin I and the Phe4-Ala5 and Tyr10-Glu11 bonds of amyloid beta-protein. Myonase is completely inhibited by such serine proteinase inhibitors as chymostatin, diisopropylfluorophosphate and phenylmethylsulfonyl fluoride, but not by p-tosyl-L-phenylalanine chloromethyl ketone, p-tosyl-L-lysine chloromethyl ketone, pepstatin, E-64, EDTA, and o-phenanthroline. It is also inhibited by lima bean trypsin inhibitor, soy bean trypsin inhibitor, and human plasma alpha1-antichymotrysin. These properties match those of chymase, but unlike chymase, myonase does not interact with heparin in the regulation of its activity. Myonase was immunohistochemically localized in myocytes, but not in mast cells.     

Infection with Chlamydia trachomatis alters the tyrosine phosphorylation and/or localization of several host cell proteins including cortactin

Infection of epithelial cells by two biovars of Chlamydia trachomatis results in the tyrosine phosphorylation of several host proteins. The most prominent change in host protein tyrosine phosphorylation involves a complex of proteins with molecular masses of 75 to 85 kDa (pp75/85) and 100 kDa (pp100). The C. trachomatis-induced tyrosine phosphorylation of pp75/85 and pp100 is observed in several cell lines, including epithelial cells, fibroblasts, and macrophages. Subcellular fractionation and detergent solubility properties of pp75/85 are consistent with its association with the cytoskeleton. Phosphoamino acid analysis demonstrates that the pp75/85 complex is phosphorylated on both tyrosine and serine residues. Immunofluorescence studies of chlamydia-infected cells by using fluorescein isothiocyanate-phalloidin and antibodies to phosphotyrosine and cortactin demonstrate that tyrosine-phosphorylated proteins, as well as cortactin, are localized to the chlamydial vacuole and that this process is facilitated by actin.     

Isolation, characterisation and cell growth-regulatory properties of kumara (sweet potato) trypsin inhibitors

Locally grown kumara (sweet potato; Ipomea batatis) was used as the starting point for the purification of a proteinase inhibitor. The purified inhibitor was highly specific for trypsin, and much less effective as an inhibitor of chymotrypsin. Two 22kDa variants were present, closely homologous to each other and to sporamin A, but with a single amino acid substitution (proline in place of serine, the second residue in mature sporamin A). One variant had the same N-terminus as sporamin A, whereas the other had a tripeptide N-terminal extension, which may represent an intermediate in the proteolytic processing of the precursor protein. A larger variant was apparently a disulphide-linked dimer of the monomeric inhibitor. A rabbit polyclonal antiserum prepared against the trypsin inhibitor reacted with all of these variants, but did not cross-react with commercially-available soybean proteinase inhibitors. The purified inhibitor did resemble other proteinase inhibitors in having a biphasic effect upon the proliferation of human fibroblasts, with a mitogenic action at low concentrations, and an inhibitory effect at higher concentrations.     

Impairment of Fas-antigen expression in adriamycin-resistant but not TNF-resistant MCF7 tumor cells

The proteinase inhibitor set in skeletal muscle is poorly characterized at present. This study was aimed to investigate in mouse skeletal muscle 1) the tissue-associated counterpart, if any, of serum protease inhibitors (which may also play antiproteolytic functions in tissues) and 2) calpastatin, a tissue inhibitor of calcium-activated neutral proteases (calpains). Triton-extracts were prepared from muscle homogenates of mice, which had been perfused extensively with phosphate buffered saline (PBS) (under deep anesthesia) to remove blood inhibitors. Among various inhibitors tested, the following muscle-associated inhibitors were identified by western-blotting: alpha-2-macroglobulin (185, 165, 35 kDa), alpha-1-antitrypsin (52 kDa), inter-alpha-trypsin inhibitor (220, 180 kDa) and calpastatin (70 kDa). Combined light microscope and confocal immunohistochemical experiments revealed that, in all muscles examined (soleus, plantaris, extensor digitorum longus) the above specific immunoreactivities were localized outside the muscle fibers (in periendomysium, blood vessel wall) as well as within them. Inter-alpha-trypsin inhibitor, however, completely lacked the intracellular localization. This wide distribution of proteinase inhibitors suggests that numerous muscular structures may be normally protected from unwanted proteolysis, thus providing an essential background for further studies on pathological models with altered proteolysis (m. dystrophy, denervation atrophy, etc.).     

Isolation and characterization of an intracellular serine proteinase inhibitor from a monkey kidney epithelial cell line

A recent report described a thrombin inhibitory activity in the soluble fraction of human placenta and the cytosolic fraction of K562 cells. Isolation and characterization of the functionally inactive 35-38-kDa placental form of this protein revealed that it was a novel serine proteinase inhibitor (Coughlin, P. B., Tetaz, T., and Salem, H. H. (1993) J. Biol. Chem. 268, 9541-9547). In the present study, we observed a 67-kDa sodium dodecyl sulfate (SDS)-stable complex when 125I-thrombin was incubated with the cytosolic fraction of a monkey kidney epithelial cell line, BSC-1. This complex was not observed in either the particulate cell fraction extracted with 0.2% Triton X-100 or medium conditioned by cells, suggesting that the thrombin-complexing factor is confined to the cytoplasm. The cytoplasmic antithrombin activity was purified to apparent homogeneity from the cytosol of BSC-1 cells previously pulsed with [35S]methionine by a combination of heparin-agarose chromatography, Mono Q fast protein liquid chromatography, and anhydrotrypsin-Affi-Gel 10 affinity chromatography. Analysis of the affinity-purified preparation by SDS-polyacrylamide gel electrophoresis and fluorography revealed a single protein with an apparent molecular mass of 38 kDa. The purified 38-kDa protein inhibited the amidolytic activities of thrombin, trypsin, urokinase, and factor Xa but not that of elastase. Incubation of the 38-kDa protein with excess thrombin identified approximately 60% of the labeled 38-kDa protein in an SDS-stable 67-kDa complex. The purified 38-kDa inhibitor was cleaved with cyanogen bromide and the isolated peptides subjected to microsequencing. Amino acid sequence obtained for a region within this protein exhibited significant homology with human antithrombin III and plasminogen activator inhibitors 1 and 2. This homologous peptide contained the full complement of residues designated as highly conserved in helix F of the greater serine proteinase inhibitor superfamily. In addition, an internal sequence of GGGGDIHQGF was found in the monkey cytoplasmic inhibitor, which is identical to that reported for an internal sequence of the human placental inhibitor. These findings confirm the existence of a novel cytoplasmic serine proteinase inhibitor in mammalian cells and provide additional details of its molecular properties. The physiological function of this novel serine proteinase inhibitor in cytoplasm is unknown.     

Purification of 2,3-dihydroxybiphenyl 1,2-dioxygenase from Pseudomonas putida OU83 and characterization of the gene (bphC)

The 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DBPD) of Pseudomonas putida OU83 was constitutively expressed and purified to apparent homogeneity. The apparent molecular mass of the native enzyme was 256 kDa, and the subunit molecular mass was 32 kDa. The data suggested that 2,3-DBPD was an octamer of identical subunits. The nucleotide sequence of a DNA fragment containing the bphC region was determined. The deduced protein sequence for 2,3-DBPD consisted of 292 amino acid residues, with a calculated molecular mass of 31.9 kDa, which was in agreement with data for the purified 2,3-DBPD. Nucleotide and amino acid sequence analyses of the bphC gene and its product, respectively, revealed that there was a high degree of homology between the OU83 bphC gene and the bphC genes of Pseudomonas cepacia LB400 and Pseudomonas pseudoalcaligenes KF707.     

Control of mucin molecular forms expression by salivary protease: differences with caries

1. A protease activity capable of degradation of the high mol. wt salivary mucus glycoprotein to a low mol. wt glycoprotein form was identified in human submandibular gland secretion. 2. The protease exhibited optimum activity at pH 7.0-7.4, and gave on SDS-PAGE under reducing conditions two major protein bands of 48 and 53 kDa. The enzyme showed susceptibility to PMSF, alpha 1antitrypsin, and egg white and soybean inhibitors, a characteristic typical to serine proteases. 3. The activity of the protease towards the high mol. wt mucus glycoprotein was found to be 3.8-fold higher in submandibular gland secretion of caries-resistant individuals than that of caries-susceptible. Furthermore, the enzyme from both groups displayed greater activity against the mucus glycoprotein of caries-resistant subjects. 4. Since the low mol. wt salivary mucus glycoprotein form is more efficient in bacterial clearance than the high mol. wt mucin, the enhanced expression of this indigenous salivary protease activity towards mucin may be the determining factor in the resistance to caries.     

Identification and characterization of the cytoplasmic antiproteinase (CAP) in human platelets. Evidence for the interaction of CAP with endogenous platelet proteins

To define the presence and potential role of platelet-associated protease inhibitors, we initiated a study designed to characterize the platelet components that are responsible for the formation of two SDS-stable complexes of approximately 58 and 70 kDa initially observed following the incubation of 125I-thrombin and human platelets. We demonstrate that thermal-mediated unfolding of the 58-kDa complex between 125I-thrombin and a nonsecreted platelet protein leads to an apparent molecular mass of 70 kDa. This platelet component is functionally and immunologically indistinguishable from the cytoplasmic antiproteinase (CAP), also known as placental thrombin inhibitor, a recently cloned member of the ovalbumin family of intracellular serpins (serine proteinase inhibitors). CAP-specific mRNA and antigen were detected in human platelets, suggesting that CAP synthesis occurs concurrent with platelet development. Utilizing quantitative immunoblotting, CAP antigen was estimated at 1.014 +/- 0.181 microg/10(9) nonstimulated platelets. After platelet activation with the calcium ionophore A23187, CAP antigen was detected in released microparticles at approximately 0. 195 +/- 0.031 microg/10(9) platelets and a fraction of platelet CAP was proteolytically modified. We provide evidence that these lower molecular mass species arise by cleavage of CAP at or near the reactive site loop. Most importantly, molecular sieving chromatography indicates the presence of an approximately 68-kDa SDS-labile complex between cleaved CAP and a cellular component in A23187-stimulated platelets, suggesting a physiological target of this intracellular serpin and a potential role for this inhibitor in regulating proteolytic activity that may be formed during platelet activation.     

Screening of compounds interacting with HIV-1 proteinase using optical biosensor technology

A high-resolution optical biosensor assay for screening of low-molecular-weight compounds, using an immobilized protein target, has been developed. HIV-1 proteinase was immobilized on the sensor surface by direct amine coupling and a variety of inhibitors and noninteracting reference drugs were applied to the sensor surface in a continuous flow of buffer. The procedure did not require intrinsic reporter groups, substrates, inhibitors, or other ligands for detection. By using a reference protein, the signal could be corrected for the relatively large background signal caused by differences in dimethyl sulfoxide concentration between running and sample buffers. Substances binding with high affinity (Ki in nM range) required efficient regeneration of the sensor surface and washing of the injection system between sample cycles to get consistent results. Analysis was simplified by using report points, extracted during both association and dissociation phases, and a simple graphical display of data. The optimized assay could correctly distinguish HIV-1 inhibitors from other compounds in a randomized series, indicate differences in their interaction kinetics, and reveal artifacts due to nonspecific signals, incomplete regeneration, or carryover. The method is expected to be generally applicable to secondary screening of low-molecular-weight compound libraries with proteins as targets.     

Regional cerebral blood flow increases during preparation for and processing of sensory stimuli

The transferrin receptor (TFR) has been detected in tissues characterised by a high degree of proliferation. We have developed a procedure for isolating TFR from human placental tissues by affinity chromatography on transferrin-Sepharose. Using gel filtration and electrophoresis in 7% PAAG, it has been shown that the molecular mass of the protein is 180 kDa. The protein has a subunit structure and is made up of two identical subunits, 90 kDa each. The constant for the protein binding to transferrin is equal to 5 x 10(-9) M. The yield of the protein isolated by the novel procedure exceeds 5-fold that obtained by previously described methods.