Affi-Gel Blue Chromatography Research Articles

Extra-hepatic expression of serum albumin mRNA in mouse retina

Purpose. In some mammals, serum albumin protein exists in the interphotoreceptor space (IPS), the space between photoreceptor cells and the retinal pigment epithelium. Serum albumin is synthesized largely in the liver, though low levels of extra-hepatic expression have been documented in several other tissues, including fetal rat kidney, pancreas, lung, and heart. The purpose of this study was to investigate whether serum albumin protein and mRNA are found in mouse retina. Methods. Using albumin rabbit antibodies and HRP goat anti-(rabbit IgG), we performed immunoassays on mouse IPS washes to detect the presence of serum albumin protein. Protein extracts from IPS washes were subjected to Affigel Blue chromatography. This resin has an affinity for serum albumin. Reverse transcription-polymerase chain reaction (RT-PCR) of retina total RNA was performed to search for albumin mRNA. Also, real-time reverse transcription polymerase chain reaction (RT-RT-PCR) was employed to look at the levels of expression in different age groups. Results. A constituent of the IPS washes specifically bound and eluted from Affigel Blue column, suggesting that the washes contained serum albumin. SDS PAGE revealed that the size of the constituent was 67 kDa, the size of serum albumin. This 67 kDa band reacted with mouse serum antibody. An RT-PCR amplified fragment of serum albumin mRNA from retina displayed the expected size. The sequence of this fragment is identical to authentic serum albumin cDNA sequence. RPE and choroid were negative for serum albumin mRNA. However, rd1 - /rd1 - retina was positive, suggesting that at least some serum albumin is synthesized in the inner layers of the retina. RT-RT-PCR showed that serum albumin mRNA levels in whole retina reached a maximum at about postnatal day 15 and gradually decreased to about one-sixth of maximum at 12 months age. Conclusions. Serum albumin protein and mRNA were found in mouse IPS and retina, suggesting that the protein is synthesized in the retina. The previously demonstrated ability of serum albumin to bind fatty acids and retinoids and its presence in the mouse IPS suggest a role for serum albumin in transporting retinoids in the retina or IPS, especially at young ages when concentrations appear greatest.

Immunoblot analysis of calpastatin degradation: evidence for cleavage by calpain in postmortem muscle.

A negative correlation exists between calpastatin activity and meat tenderness. Therefore, it is important to determine the mechanism of calpastatin inactivation in postmortem skeletal muscle. Western immunoblot analysis was performed to determine the protease(s) responsible for degradation of muscle calpastatin during postmortem storage. To accomplish this, purified calpastatin was digested with different proteases in vitro, and their pattern of calpastatin degradation was compared with that of calpastatin degradation in postmortem muscle. Polyclonal antibodies raised in mice against recombinant bovine skeletal muscle calpastatin were used to monitor calpastatin degradation. Lamb longissimus was stored at 4 degrees C and sampled at 0, 6, 12, 24, 72, 168, and 336 h postmortem. Postmortem storage produced a discrete pattern of calpastatin degradation products that included immunoreactive bands at approximately 100, 80, 65, 54, 32, and 29 kDa. Undegraded calpastatin (130 kDa) was barely detectable after 72 h of postmortem storage at 4 degrees C, and no immunoreactive calpastatin was observed by 336 h postmortem. For in vitro proteolysis, lamb longissimus calpastatin (0 h postmortem) was purified using Affi-Gel Blue chromatography. Calpastatin was digested with m-calpain, mu-calpain, cathepsin B, proteasome, trypsin, or chymotrypsin. Each of these enzymes degraded calpastatin. Immunoreactive fragments resulting from digestion of calpastatin with m- and mu-calpain were similar to each other and closely resembled those observed during postmortem aging of lamb longissimus at 4 degrees C. Digestion of calpastatin with mu-calpain reduced calpastatin activity. Degradation of calpastatin by other proteases resulted in unique patterns of immunoreactive fragments, distinct from that observed in longissimus. Thus, m- and(or) mu-calpain seem to be responsible for calpastatin degradation during postmortem storage of meat.

Isoelectric focusing of biotinidase and lipoamidase with the addition of non-ionic detergent

Isoelectric focusing (IEF) of purified biotinidase and lipoamidase (sialylated glycoprotein enzymes) was found to be reproducible and reliable when non-ionic detergent, Nonidet P-40 (NP-40), was added at 1% (v/v). Applications of the IEF containing 1% NP-40 on biotinidase and lipoamidase purifications were also successful; i.e. human serum biotinidase was purified by IEF after Affi-Gel Blue chromatography in the final step, and microsomal biotinidase and lipoamidase from guinea pig liver and kidney were purified by the preparative liquid IEF of Rotofor cell with 1% NP-40 followed by the final gel-permeation chromatography with Sephadex G-200. Thus, this high-recovery IEF with non-ionic detergent of NP-40 at 1% is shown to be a practical and reproducible method applicable to analysis and purification for several biotinidases and lipoamidases, and also expected to be generally applicable to the other relatively hydrophobic proteins.

Purification and physical characterization of cloned human cAMP phosphodiesterases PDE-4D and -4C.

Individual isozymes of family four cyclic-nucleotide phosphodiesterases (PDE-4s) were characterized and compared in order to advance our understanding of how PDE-4s regulate cAMP levels in cells. Full-length and shorter clones containing various functional domains were constructed and overexpressed using a recombinant baculovirus-infected Sf9 insect cell system. One form each of PDE-4C and 4D was purified 125- and 534-fold, respectively, using anion-exchange and affi-gel blue chromatography. The purified material was unaltered in size on SDS-polyacrylamide gels during purification and nearly homogeneous (> 95%) as estimated by both staining and immunoblotting. Approximately 1 mg of PDE-4D (74.7 kDa) and 3.7 mg of PDE-4C (61.4 kDa) could be isolated from a 6-L culture of cells. The physical characteristics of Stokes' radius and sedimentation coefficient for PDE-4 enzymes cloned from each of the four isogenes were determined using size-exclusion chromatography and sedimentation in glycerol gradients. Calculations indicate that both long and short forms can form dimers, although evidence for monomers and higher-order subunit association was seen. Furthermore, the results clearly show that all long and short forms of PDE-4 are highly asymmetric molecules. This work has shown that large amounts of PDE-4 proteins can be purified and characterized physically and enzymatically to yield information that will enable a greater understanding of how PDE-4 enzymes function in cells.

Albumin contamination of a purified human α1-antitrypsin preparation does not affect either structural conformation or the electrophoretic mobility of the inhibitor

A partially purified preparation of human α 1-antitrypsin ( α 1-AT) shown to be 60% active as an inhibitor of bovine trypsin, was chosen as starting material to investigate the nature and extent of contamination by human serum albumin (HSA) and to see whether or not such a contamination was responsible for both the reduced inhibitory activity and the slower migratory rate of the proteinase inhibitor in SDS-PAGE. Immunoblotting analysis revealed the presence of HSA in the unprocessed preparation of α 1-AT which, both in denaturing and non-denaturing PAGEs, had the same mobility as HSA, appearing as a single band of 65 kDa. By submitting the unprocessed α 1-AT preparation to affinity chromatography on an Affi-Gel Blue chromatography column, an apparently highly purified and homogeneous form of α 1-AT was obtained, as confirmed by measurement of inhibitory activity and densitometric scanning of SDS-PAGE in non-reducing conditions. However, immunoblotting analysis still revealed the presence of HSA in the most active fractions of the inhibitor eluted from the column, and regardless of purification degree, the molecular mass of the inhibitor was always 65 kDa. Treatment with β-mercaptoethanol led to separation in SDS-PAGE of HSA as a distinct band of about 10 kDa higher than the α 1-AT band, which instead maintained the same mobility as in non-reducing conditions. The results indicate that HSA has not been completely removed from α 1-AT, and its presence does not affect the electrophoretic mobility of the inhibitor. The possibility that the structural conformation of the α 1-AT, rather than contamination with HSA, was responsible for its abnormal slower migratory rate was therefore tested. For this purpose α 1-AT preparations of different degrees of purification were submitted to heat treatment to induce a non-inhibitory conformation such as loop-sheet polymerization. Polymerization was followed both by the appearance in SDS- and non-denaturing PAGEs of high molecular weight bands, which were mostly present in less purified preparations of the inhibitor, and by a decrease in inhibitory activity. A higher degree of polymerization with complete loss of inhibitory activity was observed in the unprocessed α 1-AT preparation when dissolved in Na-phosphate buffer at acidic pH, and after dialysis. After heat treatment, the purified α 1-AT was shown to run faster in the gel and, in both reducing and non-reducing conditions, the calculated mass of the inhibitor was that expected of about 54 kDa. After reducing treatment, high molecular weight polymers in SDS-PAGEs were reduced, strongly suggesting that disulphide bridges are also involved in the polymerization of α 1-AT. Results indicate that the mobility of α 1-AT in SDS-PAGE is crucially dependent on its structural conformation which dictates the extent of SDS binding. HSA contaminating the α 1-AT preparation does not affect the inhibitor conformation although at a higher degree of contamination and in favourable conditions, it does reduce the inhibitor activity.

Potent Antithrombin Activity and Delayed Clearance From the Circulation Characterize Recombinant Hirudin Genetically Fused to Albumin

AbstractIn this study we sought to extend the plasma half-life while maintaining the potent antithrombin activity of hirudin. We hypothesized that gene fusion of hirudin to albumin would result in the expression of a slowly cleared hirudin molecule. A hirudin variant 3 (HV3) cDNA was obtained by gene synthesis, while a 1,996-bp full-length rabbit serum albumin (RSA) cDNA was selected from a rabbit liver cDNA library. Expression of the former in COS-1 cells conferred antithrombin activity on media conditioned by the cells, while expression of the latter resulted in the secretion of a 67-kD protein that reacted with mono-specific anti-RSA antibodies. Having shown independent expression of the two proteins, we next expressed two fusion proteins: HV3 linked via its C-terminus to albumin (HLA), and HV3 linked via its N-terminus to albumin (ALH). The former, but not the latter, inhibited both the amidolytic and fibrinogenolytic activities of thrombin. HLA also retained the dye-binding characteristics of RSA, as judged by Affi-Gel Blue chromatography. Highly similar concentrations of either commercial HV1 (40 nmol/L) or HLA (30 nmol/L) were required to halve the initial rate of thrombin reaction with chromogenic substrate S2238, suggesting the retention of high-affinity inhibition of thrombin by the fusion protein. An His-tagged form of HLA was purified by Ni2+-chelate affinity and heparin-Sepharose chromatography. The purified, radioiodinated protein was injected into rabbits, and demonstrated a catabolic half-life of 4.60 ± 0.16 days. This represents an extension of hirudin half-life in vivo of greater than two orders of magnitude; gel analysis of HLA(H)6 recovered from rabbits showed that it circulated in intact form. Our results provide a rationale for future testing of the biological effects of HLA, and support our initial hypothesis.

An Extracellular Substance from the White-Rot Basidiomycete Phanerochaete chrysosporium for Reducing Molecular Oxygen and Ferric Iron

An extracellular substance that had single-electron oxidation activity was isolated from wood-containing cultures of the white-rot basidiomycete Phanerochaete chrysosporium. The substance was partially purified by acetone precipitation, gel filtration chromatography on Sephadex G-50 and G-25, and DEAE Affi-Gel Blue gel chromatography. It contained about 18% protein, 20% neutral carbohydrate and 0.28% Fe(II) by weight. This substance was low molecular weight and could catalyze redox reactions between an electron donor, such as NADH, and O 2 to produce H 2 O 2 and to reduce H 2 O 2 to OH. It reduced Fe(III) to Fe(II) and strongly absorbed Fe(II). This extracellular substance seems to be involved in the initial degradation of cellulose and lignin in wood by the fungus.

A putative new proteinous factor negative for stromal growth. Purification and identification from endometrial carcinoma extract

Uterine endometrial carcinoma has been reported to synthesize and secrete some putative mitogens that elicit either a positive or negative proliferation response in endometrial fibroblasts. The purposes of this study were to isolate and to identify the negative growth factor(s) from endometrial carcinoma extract. The factor was isolated by a sequence of molecular size exclusion filtration, anion exchange chromatography, gel filtration and Affi-Gel Blue chromatography, followed by NH2-terminal amino acid sequencing. Mitogenicity was determined by [3H]thymidine incorporation into the endometrial fibroblasts. The purification procedure yielded a single active protein band (68 kDa). The protein, purified approximately 20,000-fold, evoked 90% inhibition of [3H]-thymidine incorporation into endometrial fibroblasts in the nanomolar range. This potent growth inhibitor is a previously unidentified protein molecule as revealed by amino acid sequences. Endometrial carcinoma could produce a new protein that may act as a paracrine factor to suppress the growth of its stroma endometrial fibroblasts.

Purification and characterization of a flavin-binding storage protein from the hemolymph of Galleria mellonella.

The 85K storage protein that accumulates in the hemolymph of Galleria mellonella during the final larval instar was isolated and purified from newly molted pupae. The separation of fresh hemolymph proteins from larvae or pupae by different chromatographic and electrophoretic procedures indicated the native protein had a M(r) of 170,000 and consisted of two identical 85K subunits. Crosslinking experiments using fresh hemolymph followed by Western blotting also indicated a dimeric structure for the native protein. Analyses of the dimer purified from pupal hemolymph indicated that 85K was a glycoprotein, containing approximately 6.5% neutral sugar and about 1.9% amino sugar. Like other insect flavin-binding proteins, 85K has a relatively high histidine content but an uncharacteristically high arginine content. The purified 85K dimer did not bind riboflavin, suggesting that the integrity of the molecule had been altered during purification. However, 85K purified in low yield by Affi-Gel Blue chromatography, did bind riboflavin, indicating that under certain, undefined conditions the functional integrity of the protein could be retained during purification.

Isolation and Characterization of Sepiapterin Reductase from Drosophila melanogaster

Summary Sepiapterin reductase, an enzyme that catalyses the synthesis of tetrahydrobiopterin (BH4). was partially purified from Drosophila melanogaster using ammonium sulfate fractionation. Affi-gel blue chromatography and hydroxyapatite chromatography. The molecular weight of the enzyme determined by Ultrogel AcA44 column was 39,000. When the enzyme was subjected to polyacrylamide gel electrophoresis in SDS. a 38.000 MW species was found to be the major protein band. The Km values for sepiapterin and NADPH were determined to he 75.4 µM. and 14 µM, respectively. The optimal temperature and pH for the reaction were 30°C and pH 5.7-6.7. The half-life of the activity was 30 minutes when treated at 48°C The enzyme was markedly inhibited by tri-and tetravalent cations. Fe3+ . Sn4+ and divalent cation. Cd2+ . It was found that pyrimidodiazcpine (a homopterin) and 2.4-diamino-6.7-diisopropyl caused the reduction of sepiapterin reductase activity by about 50% at the concentration of 0.1 mM. Among the neurotransmitters and their precursors tested. 3 mM concentrations of melatonin and N-acetylserotonin inhibited the enzyme activity completely. In addition to sepiapterin. the enzyme uses rather broad spectrum of carbonyl compounds as substrate including menadione. p-nitrohenzaldehyde, and various dicarhonyl compounds

Complement-dependent lysis of tumor cells by a baboon IgM antibody to a tumor-associated antigen.

We developed a high-titer polyclonal antiserum to a glycoprotein tumor-associated antigen (TAA) by immunization of a baboon with the purified glycoprotein antigen. The baboon serum was fractionated into IgG and IgM components by DEAE Affi-Gel blue chromatography. The ability of the baboon IgM anti-TAA antibody to effect tumor cell lysis in the presence of complement was tested using a chromium-release assay. The baboon antibody was able to lyse melanoma target cells (20.8%-71.4% cytolysis), breast carcinoma cells (36.5%-38.9% cytolysis), and a neuroblastoma cell line (35.5% cytolysis) in the presence of complement but did not effect significant lysis of autologous lymphoblastoid cell lines (4.9% cytolysis) or peripheral blood lymphocytes from healthy volunteers (12.6% cytolysis). Cytolysis of melanoma target cells was completely inhibited by preabsorption of the IgM anti-TAA antibody with UCLA-SO-M14 (M14) cells and partially inhibited by preabsorption with several other melanoma cell lines. There was no significant inhibition of tumor cell lysis after preabsorption of the antibody with lymphoblastoid cell lines. Complement-dependent lysis of M14 targets could be blocked by addition of the purified antigen to the antibody prior to incubation with the tumor cells. Our results suggest that the glycoprotein TAA resides on the tumor cell surface and that the baboon IgM anti-TAA antibody recognizes the antigen on the cell surface and is able to fix complement and effect the lysis of the tumor cells.

Purification and composition of Ca2+/Mg2+ ATPase from rat heart plasma membrane

The Ca2+/Mg2+ ATPase, which is activated by millimolar concentrations of Ca2+ or Mg2+, was solubilized from rat heart plasma membrane by employing lysophosphatidylcholine, CHAPS, NaI, EDTA and Tris-HCl at pH 7.4. The enzyme was purified by sucrose density gradient, Affi-Gel Blue column and Sepharose 6B column chromatography. The purified enzyme was seen as a single peptide band in the sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular weight of about 90,000. The apparent molecular weight of the holoenzyme as determined under non-dissociating conditions by gel filtration on Sepharose 6B column was about 180,000 indicating two subunits. The enzyme was insensitive to ouabain, verapamil, vanadate, oligomycin, N,N-dicyclohexylcarbodiimide and NaN3, but was markedly inhibited by 20 microM gramicidin S and 50 microM trifluoperazine. Analysis of the purified Ca2+/Mg2+ ATPase revealed the presence of 17 amino acids where leucine, glutamic acid and aspartic acid were the major components and histidine, cysteine and methionine were the minor components. The purified enzyme was associated with 19.7 mumol phospholipid/mg protein which was 60 times higher than the phospholipid content in plasma membrane. The cholesterol content in the purified enzyme preparation was 0.75 mumol/mg protein and this represented an 8-fold enrichment over plasma membrane. The glycoprotein nature of the enzyme was evident from the positive periodic acid-Schiff staining of the purified Ca2+/Mg2+ ATPase in the sodium dodecyl sulfate polyacrylamide gel. The polysaccharide content of the enzyme was enriched 8-fold over plasma membrane; neuraminidase treatment decreased the polysaccharide content. Concanavalin A prevented the ATP-dependent inactivation of the purified Ca2+/Mg2+ ATPase and was found to bind to the purified enzyme with a KD of 576 nM and Bmax of 4.52 nmol/mg protein. The results indicate that Ca2+/Mg2+ ATPase is a glycoprotein and contains a large amount of lipids.

51] Purification of guinea pig uterus phosphoinositide-specific phospholipase C

Publisher Summary This chapter presents a study on the purification process of guinea pig uterus phosphoinositide-specific phospholipase C. Uteruses are collected from mature (400–600 g) asynchronous guinea pigs and washed in ice-cold phosphate-buffered saline. Fat and connective tissue are removed and no attempt is made to selectively isolate endometrium or myometrium. Uteruses are homogenized in 10 mM Tris-HCl (pH 7.4), 5 mM MgC12 , 1 mM ethylenediaminetetraacetic acid (EDTA), 0.25 M sucrose, 0.5 mM phenylmethylsulfonyl fluoride, 0.1 mM leupeptin, and 10 μg/ml aprotinin with a Polytron homogenizer. The homogenate is centrifuged at 1000 g for 15 min and the subsequent supernatant is centrifuged at 15,000 g for 20 min, then at 100,000 g for 60 min. Heparin-Sepharose fractions containing phosphoinositide-specific phospholipase C (PI-PLC) enzyme activity are pooled, concentrated to 2 ml by an Amicon concentrator, and applied to a Sephacryl S-200 column equilibrated with 20 mM Tris-HCl (pH 7.0), 75 mM KCl, 0.2 mM ethylene glycol tetraacetic acid (EGTA), and 20% glycerol at a flow rate of 20 ml/hr. Both PI-PLC Ia and Ib elute from the Sephacryl S-200 column with an apparent molecular weight of 58,000, while PI-PLC II elutes from the column at a position corresponding to a molecular weight of 72,000. Following Sephacryl S-200 chromatography PI-PLC Ib appears to be 80% pure, whereas PI-PLC Ia is approximately 50% pure. Further purification of PI-PLC Ib to greater than 95% purity is achieved by step elution from an Affi-Gel blue column. Following Affi-Gel blue chromatography, PI-PLC Ib is purified greater than 1400-fold, giving a final yield of 3.6%. Neither PI-PLC Ia nor PI-PLC II are purified to homogeneity. Antibodies prepared against PI-PLC Ib cross-react with a 60K protein in the PI-PLC Ia fractions, suggesting that PI-PLC Ia and Ib are immunologically related.

Purification of transferrins and lactoferrin using DEAE Affi-Gel blue

1.1. A simple method for purifying transferrins and lactoferrin is described.2.2. The method consists of a preliminary step ofdye-ligand chromatography using DEAE Affi-Gel Blue as the gel matrix at pH 7.5. In this chromatographic step, the transferrins and lactoferrin were readily separated from the bulk of the other proteins by start buffer elution.3.3. Differences in the chromatographic behaviour of the various serum transferrins (monkey, human, rabbit, pig, chicken and duck) and ovotransferrin upon DEAE Affi-Gel Blue chromotography can be attributed to differences in the anionic charge of the transferrins in 0.02 M potassium phosphate buffer, pH 7.5, thus resulting in the differential retardation of these protein molecules by the gel matrix.4.4. The result of DEAE Affi-Gel Blue chromatography of human lactoferrin is different from that for the transferrins. This may possibly reflect the differences in the strength of interaction between lactoferrin and transferrin with this gel matrix.

Isolation, partial purification and characterization of nuclear retinoic acid receptors from chick skin

Nuclear receptors (RARs) for retinoic acid (RA) are considered to be the ultimate mediators of the action of RA in the control of cell differentiation and inhibition of tumorigenesis. We have isolated and partially purified and characterized RAR from a RA-responsive tissue, chick embryo skin. The purification steps included Affi-Gel blue chromatography, ultrafiltration, size exclusion chromatography, and preparative isoelectric focusing. The electrofocusing of RAR-[ 3H]RA complex in ampholines (pH 3–10) revealed that the receptors have an isoelectric pH of 7.5. Whereas pronase-digested the RAR-[ 3H]RA complex completely, DNase showed 20–35% and RNase showed negligible digestive action on the complex. The ligand binding to RAR was completely inhibited by a mercury compound. RAR-α- and RAR-β-specific antibodies, on Western blot analysis, immunoreacted with a protein having a molecular weight of 50,000, presumably RAR. Binding affinity studies revealed that biologically active analogs of RA with a free COOH group (e.g., 13- cis-RA, RO-13-7410, Ch 55, and Am 80) showed, like RA, high binding affinity for RAR, whereas biologically ineffective analogs of RA (e.g., furyl and pyridyl) were poor binders. Other groups of retinoids, in which the COOH group was either lacking or blocked, did not bind to RAR whether or not they were biologically active.

Increased level of paired helical filament (PHF) antigens in CSF from patients with subacute sclerosinc panencephalitis (SSPE) : ∗P.D. Mehta 1, S.P. Mehta 1, W. Sobczyk 2, J. Kulczycki 2 and H.M. Wisniewski 1. NY State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314 USA 1 and Psychoneurological Institute, Warsaw, Poland 2

A ∼40-residue fragment of the β-amyloid precursor protein (APP) is progressively deposited in the extracellular spaces of brain and blood vessels in Alzheimer's disease (AD), Down's syndrome and aged normal subjects. Soluble, truncated forms of APP lacking the carboxyl terminus are normally secreted from cultured cells expressing this protein and are found in cerebrospinal fluid. Here, we report the detection of a similar soluble APP isoform in human plasma. This ∼125 kDa protein, which was isolated from plasma by Affi-Gel Blue chromatography or dialysis-induced precipitation, comigrates with the larger of the two major soluble APP forms present in spinal fluid and contains the Kunitz protease inhibitor insert. It thus derives from the APP751 and APP770 precursors; a soluble form of APP695 has not yet been detected in plasma. The ∼125 kDa plasma form lacks the C-terminal region and is unlikely to serve as a precursor for the β-protein that forms the amyloid in AD.

Detection of soluble forms of the β-amyloid precursor protein in human plasma

A ∼40-residue fragment of the β-amyloid precursor protein (APP) is progressively deposited in the extracellular spaces of brain and blood vessels in Alzheimer's disease (AD), Down's syndrome and aged normal subjects. Soluble, truncated forms of APP lacking the carboxyl terminus are normally secreted from cultured cells expressing this protein and are found in cerebrospinal fluid. Here, we report the detection of a similar soluble APP isoform in human plasma. This ∼125 kDa protein, which was isolated from plasma by Affi-Gel Blue chromatography or dialysis-induced precipitation, comigrates with the larger of the two major soluble APP forms present in spinal fluid and contains the Kunitz protease inhibitor insert. It thus derives from the APP751 and APP770 precursors; a soluble form of APP695 has not yet been detected in plasma. The ∼125 kDa plasma form lacks the C-terminal region and is unlikely to serve as a precursor for the β-protein that forms the amyloid in AD.

Purification and analysis of the antigenicity of a 69000 Da protein from Bordetella pertussis

A purification scheme was devised for a 69-kDa outer membrane protein of Bordetella pertussis, a virulence-associated protein which may play a role in the pathogenesis of the organism. The protein was purified to apparent homogeneity by heating B. pertussis cells for 1 h at 60°C followed by DEAE-Sepharose and Affi-Gel Blue chromatography. Antibodies found in sera obtained from patients diagnosed as having pertussis reacted with this protein. This purification scheme should be useful for the production of the 69 kDa protein which is currently being evaluated as a pertussis vaccine candidate.

Partial purification and characterization of human megakaryocyte colony-stimulating factor (Meg-CSF)

Megakaryocyte colony-stimulating factor (Meg-CSF) in urinary extracts from patients with aplastic anemia was partially characterized and purified. Using Meg-CSF-enriched fractions, we established that the moiety has the following characteristics: 1) portions of the molecules having Meg-CSF activity have sialic acid, probably with a biantennary structure, and beta-galactose residues as the terminal and penultimate sugars; 2) disulfide residues are an essential chemical group of the molecule and are located on its surface; and 3) Meg-CSF activity is stable in n-propanol, but not in acetonitrile with trifluoroacetic acid. Partial purification of Meg-CSF by a four-step procedure of ethanol precipitation, CM Affi-Gel Blue chromatography, wheat germ agglutinin-sepharose chromatography, and high-resolution hydroxyapatite chromatography, yielded a concentrate with a 430- to 630-fold increase in specific activity. The partially purified Meg-CSF fractions stimulated both human and murine megakaryocytopoiesis in vitro (CFU-meg). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreduced conditions, Meg-CSF activity was recovered in the 29-34 kDa molecular weight fractions. We have also shown that Meg-CSF, purified from the urine of aplastic anemia patients, stimulated murine megakaryocytopoiesis and platelet production in vivo. Final purification of human urinary Meg-CSF is currently in progress.

Resolution of soluble cyclic nucleotide phosphodiesterase isoenzymes, from liver and hepatocytes, identifies a novel IBMX-insensitive form

DEAE chromatography of a high speed supernatant fraction from a homogenate of rat liver, prepared under isotonic conditions in the presence of protease inhibitors, yielded three peaks of cyclic nucleotide phosphodiesterase activity (PDE activity). The first peak could be resolved on Affi-gel Blue chromatography to yield a Ca 2+/calmodulin stimulated cyclic GMP specific PDE and a cyclic AMP and cyclic GMP hydrolysing PDE whose activity was insensitive to Ca 2+/calmodulin. These two activities could also be clearly resolved by Mono-Q chromatography of soluble extracts from both liver and hepatocytes. These had different molecular weights, kinetics of substrate utilization, thermostabilities, dependence on Mg 2+ and inhibitor sensitivities. The cyclic AMP and cyclic GMP utilizing PDE resolved in these procedures appears to be a novel enzyme form (PDE-MQ-I) which is insensitive to inhibition by the so-called non-selective PDE inhibitor IBMX and displays catalytic activity in the absence of Mg 2+. None of the inhibitors tested were capable of inhibiting this form showing that the catalytic activity of this species could be distinguished from all the other soluble activities. This novel enzyme hydrolysed both cyclic AMP and cyclic GMP with K m values of 25 μM and 237 μM, respectively. The V max ratio of hydrolysis of cyclic GMP/cyclic AMP was above unity (1.4). It accounted for 30% of the soluble cyclic AMP PDE activity and 10% of the cyclic GMP PDE activity assessed at 1 μM substrate. Gel filtration of PDE-MQ-I indicated a size of 33,150 Da, in contrast to the size of 237,500 Da observed for the Ca 2+/calmodulin PDE-MQ-II. Thermal inactivation of PDE-MQ-I and PDE-MQ-II yielded single exponential decays with t 1 2 values of 6.33 min and 0.7 min at 60° respectively. In the presence of saturating Ca 2+, PDE-MQ-II was activated by calmodulin with an EC 50 of ca. 30 ng/ml. In the presence of calmodulin, PDE-MQ-II was activated by Ca 2+ with an EC 50 of ca. 20 μM. Chromatography of homogenates on Mono-Q also identified a cyclic GMP-activated cyclic nucleotide PDE (PDE-MQ-III) and two cyclic AMP specific activities (PDE-MQ-IV and PDE-MQ-V). These exhibited very different inhibitor sensitivities and could be readily distinguished using the compound Ro-20-1724 which yielded IC 50 values for inhibition of >500 μM, 13 μM and 1.5 μM, respectively, for the hepatocyte enzymes. PDE-MQ-III to -V exhibited K m values for the hydrolysis of cyclic AMP of 37.7 μM, 0.62 μM and 0.62 μM and Hill coefficients of 1.62, 0.69 and 1.01, respectively. Rolipram was found to potently inhibit both the cyclic AMP specific forms. The compound ICI 118233 failed to inhibit any of the isolated soluble phosphodiesterases. A similar multiplicity of phosphodiesterase species was observed in the soluble fraction from both rat liver homogenates and isolated hepatocytes. We suggest that care be taken in interpreting experiments performed to assess adenylate cyclase activity in intact cells by monitoring intracellular cyclic AMP accumulation in the presence of IBMX due to the possible presence of an IBMX-insensitive phosphodiesterase.