Purification and subunit structure of hepatocyte growth factor from rat platelets

A hepatocyte growth factor (HGF) that stimulates DNA synthesis of adult rat hepatocytes in primary culture was purified as a homogeneous material from platelets of 1000 rats by a four-step procedure: stimulation of its release from platelets by thrombin, cation-exchanger fast protein liquid chromatography (FPLC) on a Mono S column, heparin-Sepharose CL-6B chromatography, and reverse-phase HPLC on a C4 column. The purified HGF stimulated DNA synthesis of adult rat hepatocytes in primary culture at 1 ng/ml and was maximally effective at 5 ng/ml, being about twice as potent as EGF at this concentration. HGF did not stimulate DNA synthesis of Swiss 3T3 cells. It was found to be a heat- and acid-labile protein that was inactivated by reduction with dithiothreitol. The purified HGF had a molecular mass of 82 kDa, as estimated by SDS-PAGE, and was found to be a heterodimer which dissociated into a large subunit of 69 kDa and a small one of 34 kDa by SDS-PAGE under reducing conditions. These biological and chemical properties showed that HGF was not identical with any known growth factors, including platelet-derived growth factor (PDGF).     

Purification and properties of thermostable xylanase and beta-xylosidase produced by a newly isolated Bacillus stearothermophilus strain.

We isolated a thermophilic bacterium that produces both xylanase and beta-xylosidase. Based on taxonomical research, this bacterium was identified as Bacillus stearothermophilus. Each extracellular enzyme was separated by hydrophobic chromatography by using a Toyopearl HW-65 column, followed by gel filtration with a Sephacryl S-200 column. Each enzyme in the culture was further purified to homogeneity (62-fold for xylanase and 72-fold for beta-xylosidase) by using a fast protein liquid chromatography system with a Mono Q HR 5/5 column. The optimum temperatures were 60 degrees C for xylanase and 70 degrees C for beta-xylosidase. The isoelectric points and molecular masses were 5.1 and 39.5 kDa for xylanase and 4.2 and 150 kDa for beta-xylosidase, respectively. Heat treatment at 60 degrees C for 1 h did not cause inhibition of the activities of these enzymes. The action of the two enzymes on xylan gave only xylose.     

Purification to apparent homogeneity of a factor stimulating the growth of multiple lineages of hemopoietic cells

A glycoprotein that stimulates the proliferation of multiple hemopoietic stem and progenitor cell types was purified to apparent homogeneity. The factor, termed P cell-stimulating factor (PSF), was assayed by its ability to support the growth of murine factor-dependent hemopoietic cell lines operationally termed persisting cells (P cells). PSF was purified 50,000-fold from serum-free medium conditioned by the myelomonocytic cell line WEHI-3B by sequential ammonium sulfate precipitation, phenyl boronate chromatography, gel filtration on Sephadex G-100, neuraminidase treatment, Mono Q anion exchange chromatography, reverse phase high performance liquid chromatography on a C18 silica column, and two steps of high performance gel permeation chromatography on a TSK 3000 SW column operated under first neutral and then acidic solvent conditions. Although purified PSF could not be detected on sodium dodecyl sulfate-polyacrylamide gels stained with silver, following electrophoresis of purified PSF labeled with iodine-125, autoradiography showed only a single broad band of Mr = 30,000. This labeled band corresponded to the profile of PSF activity eluted from polyacrylamide gel slices. After reduction, labeled PSF had a slightly higher Mr of 32,000, although reduction resulted in loss of 98% of PSF activity, thus suggesting that the integrity of internal disulfide bond(s) was required for activity. When purified PSF was chromatographed on a TSK 3000 SW column under denaturing conditions in 0.1% sodium dodecyl sulfate, the single peak of absorbance at 280 nm coincided with a sharp peak of biological activity. The following unique NH2-terminal amino acid sequence of the purified PSF was obtained: NH2ALA -SER-Ile-Ser-X-X-Asp-Thr-His-Arg-Leu-Thr-Arg-. The concentration of PSF required for half-maximal stimulation of P cell growth was estimated as 1.3 X 10(-13) M or 4 pg/ml. The availability of purified PSF will allow rigorous examination of the hypothesis that a single molecule acts on multiple hemopoietic cell lineages.     

Small molecular weight GTP-binding proteins in human erythrocyte ghosts

GTP-binding proteins (G proteins) were extracted from human erythrocyte ghosts by sodium cholate and purified by gel filtration on an Ultrogel AcA-44 column followed by hydroxyapatite column chromatography. At least two peaks of G proteins were separated by hydroxyapatite column chromatography. The second peak contained G proteins recognized by the antibodies against the respective alpha subunits of Gs and Gi, and the ras protein, while the G protein of the first peak was not recognized by any of these antibodies. The G protein of the first peak was purified further by Mono Q HR5/5 column chromatography. The purified G protein showed a molecular weight of about 22 kDa on SDS-polyacrylamide gel electrophoresis. This G protein (22K G) specifically bound guanosine 5'-(3-O-thio) triphosphate (GTP gamma S), GTP and GDP with a Kd value for GTP gamma S of about 50 nM. GTP gamma S-binding to 22K G was inhibited by pretreatment with N-ethylmaleimide. The G proteins recognized by the antibodies against the alpha subunit of Go and the ADP-ribosylation factor for Gs, designated as ARF, were not detected in human erythrocyte ghosts. These results indicate that there are at least two species of small molecular weight G proteins in human erythrocyte ghosts: one is the ras protein and the other is a novel G protein of 22K G.     

A guanosine diphosphatase enriched in Golgi vesicles of Saccharomyces cerevisiae. Purification and characterization

We have recently described a luminal guanosine diphosphatase activity in Golgi-like vesicles of Saccharomyces cerevisiae (Abeijon, C., Orlean, P., Robbins, P. W., and Hirschberg, C. B. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6935-6939). The presumed in vivo role of this enzyme is to convert GDP into GMP. GDP is a reaction product following outer-chain mannosylation of luminal proteins and a known inhibitor of mannosyltransferases. It is hypothesized that GMP then returns to the cytosol. We have purified this enzyme to apparent homogeneity. Following solubilization from a membrane pellet using a buffer containing Triton X-100, the enzyme was purified on a concanavalin A-Sepharose column followed by Mono Q fast protein liquid chromatography (FPLC) and Superose-12 FPLC columns. After treatment with endoglycosidase H, the deglycosylated active enzyme was applied to a second Mono Q FPLC column and a phenyl-Superose FPLC column. The final enzyme activity was enriched 6500-fold over that of the Triton X-100 extract. The apparant molecular mass of the deglycosylated enzyme is 47 kDa. The purified enzyme is highly specific for guanosine diphosphate, requires Ca2+ for maximal activity, and has a broad pH optimum between 7.4 and 8.2. The apparent Km for GDP is 0.1 mM; the Vmax is 4.9 mmol/min/mg of protein. An enzyme activity with similar substrate specificity has also been detected in membranes of Schizosaccharomyces pombe.     

Large-scale purification of plasmid DNA by fast protein liquid chromatography using a Hi-Load Q Sepharose column.

The large-scale purification of plasmid DNA was achieved using fast protein liquid chromatography on a Hi-Load Q Sepharose column. This method allows for the purification of plasmids starting from crude plasmid DNA, prepared by a simple alkaline lysis procedure, to pure DNA in less than 5 h. In contrast to the previously described plasmid purification methods of CsCl gradient centrifugation or high-pressure liquid chromatography, this method does not require the use of any hazardous or expensive chemicals. More than 100 plasmids varying in size from 3 to 15 kb have been purified using this procedure. A Mono Q Sepharose column was initially used to purify plasmids smaller than 8.0 kb; however, a Hi-Load Q Sepharose column proved more effective with plasmids larger than 8 kb. The loading of plasmids larger than 8 kb on the Mono Q column resulted in a high back pressure and the plasmid DNA could not be eluted from the column. Thus, for routine purification we utilize the Hi-Load Q Sepharose column. Plasmids purified by this method had purity, yield, and transfection efficiency in mammalian cells similar to those of plasmids purified by CsCl density gradient centrifugation.     

Purification and characterization of nitric oxide synthase from Staphylococcus aureus

We previously reported the presence of nitric oxide synthase (NOS) in Staphylococcus aureus ATCC6538P whose activity was induced by methanol. In the present study, the methanol-induced NOS was purified 900-fold from S. aureus by means of Mono Q ion exchange column, 2',5'-ADP-agarose affinity column, and Superdex 200HR gel permeation column chromatography. The purified bacterial NOS showed two protein bands with 67 and 64 kDa molecular mass on SDS-PAGE. However, the molecular mass of the NOS was 135 kDa on Superdex 200HR gel permeation column chromatography, indicating that the native enzyme exists as a heterodimer. This bacterial NOS had K(m) value of 13.4x10(-6) M for L-arginine and V(max) of 35.3 nmol min(-1) mg(-1) protein. In addition, reduced nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, flavin mononucleotide, tetrahydrobiopterin, calmodulin and Ca(2+) were required as cofactors in the conversion of L-arginine to L-citrulline, and NOS inhibitors selectively inhibited the activity of the purified NOS.     

Purification and characterization of two soluble acid invertase isozymes from Japanese pear fruit

Two isozymes (AIV I and AIV II) of soluble acid invertase (EC 3.2.1.26) were purified from Japanese pear fruit through procedures including (NH(4))(2)SO(4) precipitating, DEAE-Sephacel column chromatography, Concanavalin A (ConA)-Sepharose affinity chromatography, hydroxyapatite column chromatography and Mono Q HR 5/5 column chromatography. The specific activities of purified AIV I and AIV II were 2670 and 2340 (nkat/mg protein), respectively. AIV I was a monomeric enzyme of 80 kDa, while AIV II may be also a monomeric enzyme, which is easy to be cleaved to 52 kDa and 34 kDa polypeptide during preparation by SDS-PAGE. The Km values for sucrose of AIV I and AIV II were 3.33 and 4.58 mM, respectively, and optimum pH of both enzyme activities was pH 4.5.     

Tandem translation of E. coli initiation factor IF2 beta: purification and characterization in vitro of two active forms.

Two forms of E. coli initiation factor IF2, IF2 alpha and IF2 beta, have been known for several years. Both forms are products of the gene infB with translational initiation at codon 1 (AUG) and codon 158 (GUG) in the same reading frame. In this work we demonstrate that IF2 beta exists in two forms, IF2 beta and IF2 beta' with initiation codons 158 (GUG) and 165 (AUG) and molecular masses of 79.7 kDa and 78.8 kDa respectively. We have recently described a fast purification method for IF2 alpha, using an FPLC procedure consisting of ion-exchange liquid chromatography on Q Sepharose HP, Mono Q and Mono S. After the Mono Q step, an apparently homogeneous IF2 beta was observed when analyzed by SDS-PAGE. However the chromatography on Mono S results in the elution of two peaks containing IF2 beta. The N-terminal amino acid sequence of the two proteins identified the first peak to be IF2 beta and the second as a protein which we term IF2 beta' starting seven residues downstream at the AUG codon 165. The activity in vitro of the two purified forms of IF2 beta was tested by measuring the stimulation of binding of the initiator fMet-tRNA(fMet) to 70S ribosomes in the presence of GTP and poly(A,U,G) as messenger-RNA. In this assay no difference in activity is detected.     

Purification and characterization of early pregnancy factor from human pregnancy sera

Early pregnancy factor (EPF) is a pregnancy-associated protein detected in the maternal serum by using the rosette inhibition assay and by evaluating the suppression of adoptive transfer of contact sensitivity. Because of its inhibitory effect on the functional reactivity of immunocompetent cells, EPF is thought to be involved in immunoregulation of the maternal immune system during early pregnancy. EPF was purified six million-fold from the serum of pregnant women between 5 and 12 weeks of gestation. The specific activity of purified EPF was approximately 8 x 10(8) units/mg. The purification scheme involved sequential DEAE-cellulose chromatography, S-Sepharose chromatography, concanavalin A-Sepharose chromatography, heparin-Sepharose chromatography, Mono S fast protein liquid chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein has an apparent molecular weight of 21,500 as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 28,000 by gel permeation high pressure liquid chromatography. The isoelectric point of purified EPF moiety is 6.5. The biological activity was susceptible to the proteolytic enzyme trypsin, acidic pH conditions, organic solvents, and sodium dodecyl sulfate, but stable to heat treatment at 56 degrees C for 30 min and the reducing agent dithiothreitol. The biological and physicochemical properties of EPF appear to be distinct from other pregnancy-associated and immunoregulatory proteins.     

Purification and partial amino acid sequences of a phospholipase C-associated GTP-binding protein from calf thymocytes

We recently identified a phosphoinositide-specific phospholipase C (PI-PLC)-stimulating GTP-binding protein (G protein) in calf thymocyte cytosol (Wang, P., Toyoshima, S., & Osawa, T. (1987) J. Biochem. 102, 1275-1287; and (1988) 103, 137-142). In this study we completely purified a G protein whose properties are quite similar to the G protein mentioned above from the calf thymocyte membrane and determined partial amino acid sequences of it. The purification was achieved by first treating the membrane with GTP gamma S, followed by sequential column chromatographies on DEAE-Sepharose CL-6B, Sephacryl S-200, Mono Q, and Mono S. The G protein was purified in a GTP gamma S-binding form and assayed as to the radioactivity of the [35S]GTP gamma S-bound PI-PLC-associated G protein standard obtained from calf thymocyte cytosol. The purified G protein could stimulate the activity of a partially purified PI-PLC for phosphatidylinositol 4,5-bisphosphate hydrolysis. From approximately 5.6 g of membrane protein we obtained about 5 micrograms of a purified sample. The purified G protein showed a molecular weight of 21 kDa on SDS-PAGE and one of 25 kDa on gel filtration. The partial amino acid sequences were determined by treating the purified sample with lysylendopeptidase, purifying the resultant peptide fragments on a HPLC-reverse phase column and then sequencing the peptide fragments with a sequencer. Comparison of the obtained sequences with those of known lower molecular weight GTP-binding proteins suggested that, although structurally similar to rho gene products, this is a novel G protein.     

Study of Inhibitors of Proteinases in the Midgut Anterior Part of the Cockroach Nauphoeta cinerea

The study of proteinase inhibitors in the midgut of the omnivorous cockroach Nauphoeta cinerea was carried out under conditions excluding their food origin. One trypsin inhibitor of molecular mass of 8.0 kDa and three subtilisin inhibitors of molecular masses of 13.0, 8.0, and 4.5 kDa were found in the protein preparations, using Sephadex G-50 fractionation. 94% of the activity of the both inhibitor types were located in the anterior midgut part. Using a high performance liquid chromatography on Mono Q column, the preparation of trypsin inhibitor was purified 120 times. Its isoelectric point was to 4.3. The inhibitor lost a part of its activity both under acidic and, especially, under alkaline conditions and was completely inactivated at pH 10. The studied inhibitors inhibited effectively activities of trypsin-like and subtilisin-like proteinases from the cockroach posterior midgut part. The possible physiological role of the proteinase inhibitors and, particularly, their participation in regulation of digestion in the midgut of N. cinerea are discussed.     

An androgen-dependent subclone derived from a mouse mammary tumor, Shionogi carcinoma 115, secretes a heparin-binding growth factor having an apparent molecular weight of 31,000 in response to androgen.

An androgen-dependent cell line denoted SC2G is a clone of an androgen-dependent mouse mammary tumor, Shionogi Carcinoma 115. Fibroblast growth factors (FGFs), epidermal growth factor (EGF) and transforming growth factor-alpha (TGF alpha) are stimulatory for the growth of SC2G cells in the absence of androgen. This clone was found to secrete an androgen-induced growth factor mostly eluting at 1.8 M NaCl on a heparin-Sepharose column. This factor was partially purified by chromatography on two consecutive heparin-Sepharose columns followed by cation-exchanging chromatography on an S-Sepharose column from the chemically defined serum-free medium conditioned by SC2G cells in the presence of androgen. The factor was a heat- and acid-labile cationic protein that was inactivated by reduction with dithiothreitol. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, most of the growth-promoting activity of this factor was found at approx. 31 kDa under non-reduced conditions. Neither neutralizing antibody against basic-FGF nor that against EGF inhibited the growth-promoting activity of this factor in cell culture, suggesting the factor was distinct from basic FGF or EGF. However, the possibility that the factor was another FGF- or EGF-like growth factor was not excluded.     

Simultaneous isolation of protein C activator,fibrin clot promoting enzyme (fiprozyme) and phospholipase A2 from the venom of the southern copperhead snake

The simultaneous isolation of three enzymes from the southern copperhead snake venom (Agkistrodon contortrix contortrix; ACC) is described. The first step is a chromatography of crude venom on a Mono S cation-exchange column at pH 6.5. A fibrin clot promoting enzyme (fiprozyme) that preferentially releases fibrinopeptide B from fibrinogen is isolated from the fraction not binding to the Mono S by a further three-step process. The procedure involves affinity chromatography on Blue Sepharose, gel chromatography on Sephacryl S-200 and metal–chelate chromatography on Chelating Sepharose. Protein C activator and phospholipase coelute from the Mono S column. They are separated by a gel chromatography on Sephacryl S-200. After this step two enzymes are obtained: a highly purified protein C activator applicable in methods for determination of functional level of protein C (a plasma regulator of hemostasis) and an electrophoretically pure enzyme with the activity of phospholipase A₂.     

Purification and characterization of protein methylase II from Helicobacter pylori

Protein methylase II (AdoMet:protein-carboxyl O-methyltransferase, EC 2.1.1.24) was identified and purified 115-fold from Helicobacter pylori through Q-Sepharose ion exchange column, AdoHcy-Sepharose 4B column, and Superdex 200 HR column chromatography using FPLC. The purified preparation showed two protein bands of about 78 kDa and 29 kDa molecular mass on SDS-PAGE. On non-denaturing gel electrophoresis, the enzyme migrated as a single band with a molecular mass of 410 kDa. In addition, MALDI-TOF-MS analysis and Superdex 200 HR column chromatography of the purified enzyme showed a major mass signal with molecular mass values of 425 kDa and 430 kDa, respectively. Therefore, the above results led us to suggest that protein methylase II purified from H. pylori is composed of four heterodimers with 425 kDa (4x(78+29)=428 kDa). This magnitude of molecular mass is unusual for protein methylases II so far reported. The enzyme has an optimal pH of 6.0, a K(m) value of 5.0x10(-6) M for S-adenosyl-L-methionine and a V(max) of 205 pmol methyl-(14)C transferred min(-1) mg(-1) protein.     

Purification of an ultraviolet-inducible, damage-specific DNA-binding protein from primate cells.

A UV-inducible, damage-specific DNA-binding (DDB) protein with high affinity for double-stranded UV-irradiated DNA has been identified recently in monkey kidney (CV-1) cells (Hirschfeld, S., Levine, A. S., Ozato, K., and Proti?, M. (1990) Mol. Cell. Biol. 10, 2041-2048). We have now purified the DDB protein from extracts of CV-1 cells using hydroxylapatite, phosphocellulose, Mono S, and DNA-affinity column chromatography. The DDB activity, either from mock-treated or UV-induced cells, is heterodisperse in column chromatography, and separation of three forms of the protein was obtained on a phosphocellulose column. Analysis of purified preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that greater than 90% of all three forms is a protein of approximately 126 kDa. The size of the native DDB protein was deduced from gel filtration and native polyacrylamide gel electrophoresis to be approximately 210 kDa, which suggests that the native DDB protein in solution is a homodimer. Preparations of partially purified DDB protein from UV-treated cells have enhanced levels of DDB activity and the protein when compared with similar preparations from mock-treated cells. This damage-recognition protein, alone or in conjunction with other subunits, may be of general importance for the initial recognition of DNA damage in mammals.     

Partial purification and characterization of masking protein for beta-type transforming growth factor from rat platelets

beta-Transforming growth factor (TGF-beta) is stored in platelets and secreted as a high molecular weight latent form associated with a carrier protein of about 440 KD. This carrier protein could be separated from TGF-beta in 1 N acetic acid and could again mask the activity of TGF-beta under neutral conditions. Therefore, it was named the masking protein of TGF-beta. The masking protein was separated from TGF-beta by gel filtration on a Sephacryl S-300 column or by anion-exchanger FPLC on a Mono Q column in the presence of 6 M urea. Partially purified masking protein from rat platelets neutralized the activity of TGF-beta dose-dependently and was effective at 0.3 microgram/ml. This masking protein could also mask the activity of human TGF-beta, suggesting that it was not species specific. The masking protein was a heat- and acid-stable protein, but was inactivated by treatment with dithiothreitol. The Physiological role of the masking protein in the mechanisms of wound healing and liver regeneration is discussed.     

Preparation of electrophoretic variants of corticosteroid-binding globulin (CBG) using liquid liquid partition chromatography

Human corticosteroid-binding globulin (CBG) was purified to homogeneity by application of three different chromatographic methods. After fractionation of pregnancy serum with ammonium sulfate the 80%-pellet was used for affinity chromatography based on tresyl activated Sepharose (Pharmacia, Uppsala, Sweden). The affinity eluate was injected into a Mono Q anion exchange column (Pharmacia). Fractions containing CBG were finally purified by liquid liquid chromatography on LiParGel 750 (Merck, Darmstadt, F.R.G.). The purified protein was characterized by IEF and PAGE. This paper describes a method for the chromatographic separation of the two variants of CBG without a loss of binding activity towards steroids for each of the two characteristic bands of this protein.     

Purification of homogeneous forms of fungal peroxygenase

Extracellular peroxygenase from the agaric fungus Agrocybe aegerita is a versatile biocatalyst that oxygenates various substrates by means of hydrogen peroxide. The enzyme is routinely produced in suspensions of soybean meal and has until now been purified by several steps of fast protein liquid chromatography (FPLC) using different ion exchangers. The final protein fraction had a molecular mass of 46 kDa but still consisted of several incompletely separated proteins with slightly differing isoelectric points (pI 5.2, 5.6, 6.1), probably representing differently glycosylated isoforms. This made it difficult to further purify the individual protein forms. Since homogeneous protein fractions are a pre-requisite for X-ray crystallography and specific structure-function studies, an appropriate FPLC procedure was developed starting with pre-purification of crude peroxygenase on SP Sepharose followed by chromatofocusing on a Mono P column and elution with a pH gradient. Three sufficiently separated main protein peaks were eluted from the Mono P column and confirmed to be distinct forms of aromatic peroxygenase with different pIs. All A. aegerita peroxygenase forms oxygenated toluene and naphthalene and no catalytic differences were observed between them. We tested also two devices for preparative isoelectric focusing (Rotofor, IsoPrime systems) for peroxygenase separation but resolution and protein recovery were not sufficient.