DEAE-Sepharose Chromatography Research Articles

An inducible acid phosphatase from the yeast Pichia pastoris: characterization of the gene and its product

To develop the budding yeast Pichia pastoris (Pp) as a model system for the study of protein secretion, we have characterized a secreted acid phosphatase (Pholp) from this yeast. Pholp can be induced 100-fold by starvation for phosphate. The enzyme was purified to homogeneity from a cell-wall extract by DEAE-Sepharose chromatography. We selected mutants that lacked extracellular phosphatase activity and the gene (PHO1) encoding Pholp was isolated from a recombinant plasmid library of Pp DNA by complementation of the mutant defect. PHOI encodes a protein of 468 amino acids (aa) with homology to repressible acid phosphatases from other yeast species. The sequence contains a 15-aa N-terminal signal sequence and six potential N-linked glycosylation sites. Antiserum to Pholp was used to show that Pholp transits the Pp secretory pathway in less than 5 min.

Mechanisms of platelet activation by a stimulatory antibody: cross-linking of a novel platelet receptor for monoclonal antibody F11 with the Fc gamma RII receptor.

The mechanisms by which a stimulatory monoclonal antibody (mAb), called mAb F11, induces granular secretion and aggregation in human platelets have been characterized. Fab fragments of mAb F11, as well as an mAb directed against the platelet Fc gamma RII receptor (mAb IV.3) were found to inhibit mAb F11-induced platelet secretion and aggregation, indicating that the mAb F11 IgG molecule interacts with the Fc gamma RII receptor through its Fc domain and with its own antigen through its Fab domain. The mAb F11 recognized two platelet proteins of 32 and 35 kDa on the platelet membrane surface, as identified by Western blot analysis. We purified both proteins from human platelet membranes using DEAE-Sepharose chromatography followed by mAb F11 affinity chromatography. When added to platelet-rich plasma, the purified proteins dose-dependently inhibited mAb F11-induced platelet aggregation. The purified protein preparation also competitively inhibited the binding of 125I-labelled mAb F11 to intact platelets. The N-terminal 26 amino acid sequences of both the 32 and 35 kDa proteins were identical and contained a single unblocked serine in the N-terminal position. When digested with N-glycanase, the 32 and 35 kDa proteins were converted into a single approximately 29 kDa protein, indicating that these two proteins are derived from the same core protein but differ in their degree of glycosylation. Internal amino acid sequence analysis of the F11 antigen provided information concerning 68 amino acids and suggested two consensus phosphorylation sites for protein kinase C (PKC). The phosphorylation by PKC of the isolated F11 antigen was observed following stimulation by phorbol 12-myristate 13-acetate. Databank analysis of the N-terminal and internal amino acid sequences of the F11 antigen indicated that the N-terminal sequence exhibited the highest degree of similarity to the variable region of the alpha-chain of human T-cell receptors (TCR). In contrast, the F11 internal sequences did not exhibit any similarity to the TCR. Our results demonstrate that the F11 antigen is a novel platelet membrane surface glycoprotein which becomes cross-linked with the Fc gamma RII receptor when platelets are activated by the stimulatory mAb F11. These mechanisms may be relevant to the production of immune thrombocytopenia by platelet-activating antibodies.

Release of the Hepatitis β Virus-associated DNA Polymerase from the Viral Particle by the Proteolytic Cleavage

Previous efforts for biochemical study of the human hepatitis B virus (HBV) DNA polymerase have been limited by its tight association with viral nucleocapsids. We report here that the soluble DNA polymerase from HBV particles was obtained by low pH treatment of the viral particles followed by incubation with small amounts of subtilisin. By these treatments, the approximately 100-kDa band in the activity gel assay was gradually converted to approximately 70 kDa, which subsequently showed reverse transcriptase activity on several exogenous templates. The single approximately 70-kDa active band, which did not show any DNA polymerase activity in endogenous reaction, was eluted through DEAE-Sepharose chromatography. These results suggest that the approximately 100-kDa protein, most likely the product of HBV Pol open reading frame, is tightly associated with viral nucleocapsids, and the approximately 70-kDa protein, the proteolytic cleavage product of approximately 100-kDa enzyme, is solubilized from viral particles as an active enzyme on exogenous templates.

Purification and characterization of neomycin phosphotransferase II from genetically modified cottonseed (Gossypium hirsutum)

Expression of the nptII gene [encoding the neomycin phosphotransferase II (NPTII) enzyme] is frequently employed as a selectable marker for plant transformation in a variety of species, including cotton. Therefore, characterization of this enzyme was considered an important part of the relatory approval process designed to assess the safety of products derived from genetically modifed plants. Because of the role of cottonseed in animal feeds and other commercial uses, a purification scheme for NPTII from cottonseed was developed employing solvent extraction, a series of precipitations, DEAE-Sepharose chromatography, and affnity chromatography which yielded a highly enriched preparation that retained enzymatic activity. Further purification was obtained by SDS-PAGE, electrotransfer to PVDF membrane, and direct sequencing of the appropriate band. NPTII derived from cottonseed was characterized by purification behavior, NH 2 -terminal sequence, SDS-PAGE, immunoblot, ELISA, and enzymatic activity and was found by these criteria to be chemically equivalent to the enzyme derived from an Escherichia coli expression system

"Prohormone thiol protease" (PTP) processing of recombinant proenkephalin.

The "prohormone thiol protease" (PTP) from adrenal medullary chromaffin granules has been demonstrated as a novel cysteine protease that converts the model enkephalin precursor, ([35S]Met)-preproenkephalin, to appropriate enkephalin related peptide products [Krieger, T. J., & Hook, V. Y. H. (1991) J. Biol. Chem. 266, 8376-8383; Kreiger, T. J., Mende-Mueller, L., & Hook, V. Y. H. (1992) J. Neurochem. 59, 26-31; Azaryan, A. V., & Hook, V. Y. H. (1994) FEBS Lett. 341, 197-202]. In this report, PTP processing of authentic proenkephalin (PE) was examined with respect to production of appropriate intermediate products, and kinetics of PE processing were assessed. Recombinant PE was obtained by high level expression in Escherichia coli, with the pET3c expression vector; PE was then purified from E. coli by DEAE-Sepharose chromatography, preparative gel electrophoresis, and reverse-phase HPLC. Authentic purified PE was confirmed by amino acid composition analyses and peptide microsequencing. In time course studies, PTP converted PE (12 microM) to intermediates of 22.5, 21.7, 12.5, and 11.0 kDa that represented NH2-terminal fragments of PE, as assessed by peptide microsequencing. Differences in molecular masses of the 22.5, 21.7, 12.5, and 11.0 kDa products reflect PTP processing of PE within the COOH-terminal region of PE, which resembles PE processing in vivo [Liston, D. L., Patey, G., Rossier, J., Verbanck, P., & Vanderhaeghen, J. (1983) Science 225, 734-737; Udenfriend, S., & Kilpatrick, D. L. (1983) Arch. Biochem. Biophys. 221, 309-314]. Products of 12.5, 11.0, and 8.5 kDa were generated by PTP cleavage between Lys-Arg at the COOH-terminus of (Met)enkephalin-Arg6-Gly7-Leu8.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of free psa isoforms, psa complex formation, and specificity of anti‐psa antibodies by hplc and page‐immunoblotting techniques

Both high performance liquid chromatographic (HPLC) and polyacrylamide gel electrophoresis-immunoblotting (PAGE-immunoblotting) procedures have been established for the study of isoforms of free prostate-specific antigen (PSA) and the complex formation between free PSA and protease inhibitors, and for the evaluation of the specificities of various anti-PSA antibodies. We found multiple isoforms of free PSA on PAGE, which were all capable of forming complexes with protease inhibitors. The same isoform pattern can be produced from the original seminal fluid. The PSA isoforms differ from each other most likely in charge because they could be converted to one band on SDS-PAGE and to a single peak by gel filtration chromatography. We found it difficult to form large quantities of PSA complex when mixing free PSA from seminal fluid with protease inhibitors, regardless of whether the free PSA or the protease inhibitors were in excess. Except for the PSA-ACT complex, which was consistently detectable by both HPLC and PAGE-immunoblotting techniques after incubation, these two procedures disagreed in their detection of PSA-A2M and PSA-AT complexes. The PSA-A2M complex was usually observable by immunoblotting techniques but barely detectable on HPLC, whereas PSA-AT was totally invisible by immunoblotting but appeared as a peak in the HPLC elution profile. Mixing free PSA with serum clearly resulted in both PSA-ACT and PSA-A2M complexes. However, more PSA-ACT than PSA-A2M was formed; the result was also confirmed by using 125I-PSA for mixing. PSA could be separated into active and inactive PSA by DEAE Sepharose chromatography with a 14-fold difference in protease activity. The difference in enzymatic activity apparently had no effect on complex formation. All the anti-PSA antibodies examined in this study reacted with PSA isoforms, PSA-ACT and PSA-A2M complexes. We conclude that it would be almost impossible to establish an assay to measure all forms of PSA in the serum and to expect to produce precise and accurate PSA values.

Homologous expression of recombinant manganese peroxidase in Phanerochaete chrysosporium

The promoter region of the glyceraldehyde-3-phosphate dehydrogenase gene (gpd) was used to drive expression of mnp1, the gene encoding Mn peroxidase isozyme 1, in primary metabolic cultures of Phanerochaete chrysosporium. A 1,100-bp fragment of the P. chrysosporium gpd promoter region was fused upstream of the mnp1 gene to construct plasmid pAGM1, which contained the Schizophyllum commune ade5 gene as a selectable marker. pAGM1 was used to transform a P. chrysosporium ade1 auxotroph to prototrophy. Ade+ transformants were screened for peroxidase activity on a solid medium containing high carbon and high nitrogen (2% glucose and 24 mM NH4 tartrate) and o-anisidine as the peroxidase substrate. Several transformants that expressed high peroxidase activities were purified and analyzed further in liquid cultures. Recombinant Mn peroxidase (rMnP) was expressed and secreted by transformant cultures on day 2 under primary metabolic growth conditions (high carbon and high nitrogen), whereas endogenous wild-type mnp genes were not expressed under these conditions. Expression of rMnP was not influenced by the level of Mn in the culture medium, as previously observed for the wild-type Mn peroxidase (wtMnP). The amount of active rMnP expressed and secreted in this system was comparable to the amount of enzyme expressed by the wild-type strain under ligninolytic conditions. rMnP was purified to homogeneity by using DEAE-Sepharose chromatography, Blue Agarose chromatography, and Mono Q column chromatography. The M(r) and absorption spectrum of rMnP were essentially identical to the M(r) and absorption spectrum of wtMnP, indicating that heme insertion, folding, and secretion were normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Selenium-containing peroxidases of germinating barley.

Germinating barley grown on an artificial medium was exposed to 75Se-selenite for 8 d. Then the leaves were homogenized and proteins were separated by means of Sephadex G-150 filtration, followed by DEAE-Sepharose chromatography. Each fraction collected was assayed for total protein, radioactivity, and peroxidase activity. In barley leaves, three protein peaks (peaks no. I, II, and III) with peroxidase activity could be separated by Sephadex G 150 filtration. Each fraction was then further separated on DEAE-Sepharose chromatography. Thus, peaks I and II were resolved by DEAE-Sepharose into one major and two minor peaks of radioactivity. However, only the major peak showed peroxidase activity. Peak III was resolved from the gel filtration on the DEAE-sepharose into one major and four minor peaks of radioactivity. The major and three of the minor radioactivity peaks contained peroxidase activity. The protein fractions were separated by polyacrylamide gel electrophoresis. The molecular weights of separated proteins were estimated by means of molecular markers, and 75Se radioactivity was evaluated by autoradiography. Thus, gel filtration peak I contained four bands with mol wts of 128, 116, 100, and 89 kDa. Of these, the 89 kDa protein contained selenium. Peak II contained three protein bands with mol wts 79.4, 59.6, and 59.9. The 59.6 band was a selenoprotein. Peak III contained four protein bands (and some very weak bands). The four major bands had mol wts of 38.6, 31.6, 30.2, and 29.2 kDa. The last mentioned band was a selenoprotein.

Identification and characterization of isoenzymes of cyclic nucleotide phosphodiesterase in human kidney and heart, and the effects of new cardiotonic agents on these isoenzymes

The present study was done to identify and characterize the isoenzymes of cyclic nucleotide phosphodiesterase (PDE) and to determine their intracellular distribution in human kidney and heart. The in vitro effects of new cardiotonic agents, namely, NSP-805 (4,5-dihydro-5-methyl-6-[4-[(2-methyl-3-oxo-1-cyclopentenyl)amino] phenyl]-3(2H)-pyridazinone), TZC-5665 (6-[4-[2-[3-(5-chloro-2-cyanophenoxy)-2-hydroxypropylamino]- 2 -methylpropylamino]phenyl]-5-methyl-4,5-dihydro-3(2H)-pyridazinone ) and its metabolites, OPC-18790 ((+/-)-6-[3-(3,4-dimethoxybenzylamino)-2 -hydroxypropoxy]-2-(1H)-quinolinone), MS-857 (4-acetyl-1-methyl-7-(4-pyridyl)-5,6,7,8-tetrahydro-3(2H)-isoquinolinone ) and E-1020 (1,2-dihydro-6-methyl-2-oxo-5-(imidazo[1,2-a]pyridin-6-yl)-3-pyridine carbonitrile hydrochloride monohydrate), on these human PDE isoenzymes were also investigated. PDE isoenzymes were separated from cytosolic and particulate fractions of homogenates of human kidney and heart by DEAE-Sepharose chromatography. PDE isoenzymes were identified by their elution characteristics, substrate specificities, sensitivities to regulation by effectors and by the use of isoenzyme-specific inhibitors. In a cytosolic fraction from kidney, Ca2+/calmodulin-dependent PDE (CaM-PDE), cyclic GMP-stimulated PDE (cGS-PDE), cyclic GMP-inhibited PDE (cGI-PDE) and two forms of cyclic AMP-specific PDE (cAMP-PDE) were resolved. One form of cAMP-PDE (cAMP-PDE alpha), which was eluted at a lower ionic strength than cGI-PDE during DEAE-Sepharose chromatography, was newly recognized in human tissues, though the other form (cAMP-PDE beta), which eluted later than cGI-PDE, had been previously isolated.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of enzymatically active Onchocerca volvulus[formula omitted] superoxide dismutase expressed in Escherichia coli

The Onchocerca volvulus superoxide dismutase was expressed in Escherichia coli, using a protocol designed to produce the native enzyme rather than a fusion protein. The recombinant O. volvulus superoxide dismutase (rOVSOD) was found in the cytosol of the disrupted bacteria and represented > 10% of the total bacterial protein. The enzyme was purified to homogeneity using DEAE-Sepharose chromatography, followed by phenyl-Sepharose chromatography. The rOVSOD was enzymatically active which was demonstrated by its reactivity with O .− 2 produced either by the xanthine-xanthine oxidase system or by stimulated eosinophils. The specific activity was determined to be 4668 U mg −1. This activity could be blocked by rabbit antiserum raised against the rOVSOD. The maximal activity was obtained upon supplementation of the bacterial growth media and enzyme buffer with copper and zinc ions. Activity characteristics in the presence of inhibitors was also characteristic of a Cu/Zn superoxide dismutase. The rOVSOD has an apparent subunit molecular mass of 16 000 in SDS-PAGE. The active enzyme behaves as a dimer of 32 kDa as determined by gel filtration.

Purification and characterization of two serine collagenolytic proteases from crab Paralithodes camtschatica

Two enzymes possessing collagenolytic activity were isolated from the hepatopancreas of crab Paralithodes camtschatica by ammonium sulfate fractionation and DEAE-Sepharose chromatography. It was shown that the specific activities of proteases A and C toward insoluble collagen were equal to 400 and 300 Mandl units/mg protein, respectively. The mol. wt of homogenous proteases A and C determined by gradient polyacrylamide gel electrophoresis in the presence of SDS and 2-mercaptoethanol were equal to 30 and 24 kDa, respectively. The isoelectric point values for the enzymes were determined as 2.5 and 2.9. Both enzymes lack carbohydrates. The amino acid compositions of two crab proteases were measured. The optimal conditions for the enzyme catalysis and the catalytic constants for collagenolytic proteases A and C with respect to Bz-Arg-pNA and Bz-Tyr-OEt have been determined. Inhibition data led to classification of the purified enzymes as serine proteases.

IDENTIFICATION, CHARACTERIZATION AND FUNCTIONAL ROLE OF PHOSPHODIESTERASE ISOZYMES IN HUMAN AIRWAY SMOOTH MUSCLE

Purpose of the Study. Theophylline, a widely used bronchodilator, has been traditionally thought to exert its effect by inhibiting phosphodiesterase (PDE), which increases cAMP levels. It has become apparent that there are actually five different PDE isozymes, each encoded by a different gene or gene family. It may be possible to selectively target the lung PDE isozyme using specific inhibitors for the pulmonary smooth muscle type of PDE. The specific type of airway smooth muscle PDE isozyme, the effect of specific PDE inhibitors, individually or additively, and regional differences in PDE enzyme types was the focus of this study. Methods. Human trachea was obtained from brain-dead subjects, processed within 24 hours by separating the muscle from surrounding tissues, and homogenized. Human lung was similarly obtained and processed, with the ultimate isolation of individual bronchial smooth muscle from several generations of bronchial diameters. PDE isozyme separation was performed by using a combination of DEAE-Sepharose and calmodulin-Sepharose affinity chromatography. Results. Trachael smooth muscle contained all five PDE isozymes. Using specific inhibitors of each PDE isozyme, inhibitors of PDE type III were >PDE Type IV which were >PDE type III, respectively, as bronchial smooth muscle relaxants. This bias toward Type III and IV PDE was found in large and small bronchial preparations. Inhibitors of PDE III and IV acted synergistically, and PDE III inhibitors assisted beta-agonist-induced relaxation. Reviewer's Comments. If the only action of theophylline is its' effect on PDE, drugs that do a better job of blocking specific PDE isozyme(s) in bronchial smooth muscle should be developed.

Heat shock protein 60 (GroEL) fromPorphyromonas gingivalis: Molecular cloning and sequence analysis of its gene and purification of the recombinant protein

Porphyromonas gingivalis is associated with human periodontal disease. We cloned and sequenced the gene for heat shock protein 60 (GroEL, HSP60) from P. gingivalis FDC381. The identified clone carried a 2.6 kb DNA fragment which contained two open reading frames (ORFs) encoding a 9.6- and a 58.4-kDa protein. The translated amino acid sequence of these ORFs showed a high degree of homology with known sequences for GroES and GroEL from several bacterial species and humans. Escherichia coli carrying this clone expressed a 65-kDa protein which was recognized by anti-Mycobacterium leprae HSP60 monoclonal antibody. We purified the 65-kDa protein by DEAE-sepharose chromatography and hydroxyapatite chromatography. This protein was immunogenic and was recognized by sera from a number of patients with periodontal disease. This immunological reactivity and the existence of molecular mimicry between the P. gingivalis GroEL and other HSP homologs may indicate an important role for this molecule in periodontal lesion.

Overexpression and simple purification of human immunodeficiency virus-1 gag epitope derived from a recombinant antigen in E. coli and its use in ELISA

To develop a test for diagnosis of human immunodeficiency virus-1 (HIV-1) exposure sensitivity, a part of the gag gene was cloned and expressed in Escherichia coli, using expression vectors containing a trp promoter. The immunoreactivity of recombinant protein was determined using HIV-1 specific antibodies in a Western blot analysis. The recombinant plasmid, pYHCgag3, gag gene was fused to the trpE′ gene linked to the hydroxylamine (HA) cleavage recognition sequence which was induced to overexpress a core antigen (gag a.a. 121–398 from plasmid BH10) as fusion protein in the form of insoluble inclusion body. Recombinant gag was purified by a simple single step purification procedure. After partial purification of inclusion bodies and subject to the HA-cleavage treatment, gag protein was further purified to homogeneity using DEAE-Sepharose chromatography. The purified core antigen offered reliable results with high sensitivity and specificity for identification of HIV-1 antibodies when tested in the enzyme-linked immunosorbent assay (ELISA). These results suggest that mass production of recombinant core antigen will provide a valuable resource to HIV-1 serodiagnostics for the screening of large groups of blood donors to prevent HIV-1 infection.

Rapid Purification of Recombinant Human Tumor Necrosis Factor Beta

A rapid and improved method for the purification of recombinant human tumor necrosis factor beta (rhTNF-β) from Escherichia coli HB 101 cells has been developed. The method utilized sequential steps of polyethylenimine (PET) and ammonium sulfate precipitation to remove most of the extraneous proteins and nucleic acids from the cell extracts. The final step of purification consisted of DEAE-Sepharose chromatography at pH 7.5 in which rhTNF-β was eluted with starting buffer. This procedure, when compared to the earlier methods of purification, is highly efficient since we could increase the overall yield of rhTNF-β and reduce the purification time considerably. The final yield that we obtained from 1 liter of fermentation broth (containing approximately 80 g of wet cells) was 40-50 mg.

Purification and Characterization of Two Membrane-Bound Serine Proteinases from Rat Liver Microsomes Active in Degradation of Cytochrome P450

Two serine proteinases capable of digesting cytochrome P4502E1 (CYP2E1) have been purified from sodium cholate solubilized rat liver microsomal membranes. After chromatography on hydroxyapatite, DEAE-Sepharose chromatography resolved the CYP2E -degrading activity into two peaks, and the two proteinases were finally purified on benzamidine-Sepharose. Both have a Mr of 32,000 on SDS-PAGE, are optimally active at pH 8, and show a susceptibility to inhibitors typical of serine proteinases. CYP2E1 degradation patterns exhibited by the proteinases are identical to each other and similar to that observed during the proteolysis of endogenous CYP2E1 in the microsomal membranes, which indicates that the proteinases can degrade CYP2E1 in its native environment. We suggest a role of these proteinases in the rapid phase of cytochrome P450 degradation in the endoplasmic reticulum.

Involvement of Porphyromonas gingivalis fimbriae in adherence to Streptococcus gordonii.

Adherence of Porphyromonas gingivalis to early plaque bacteria, such as Streptococcus gordonii, is considered an important colonization mechanism. The molecules that mediate this interspecies binding have not been determined. Fimbriae were prepared from P. gingivalis 33277 by mild agitation, ammonium sulfate precipitation and DEAE-Sepharose chromatography. In a nitrocellulose blot adherence assay, purified fimbriae inhibited S. gordonii G9B-P. gingivalis 33277 binding by up to 54%. In addition, fimbriae bound to S. gordonii cells in a dot-blot assay. Incubation of fimbriae with S. gordonii cells followed by washing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electroblotting and probing with P. gingivalis antibodies also revealed that the fimbriae bind to S. gordonii. In contrast, S. gordonii did not interact with fimbriae that were first subjected to SDS-PAGE and electroblotting or deposited on a nitrocellulose membrane, suggesting that conformational determinants of the fimbriae may be important in binding. The results indicate that binding between P. gingivalis and S. gordonii is mediated, at least in part, by the porphyromonads' fimbriae.

Isolation and characterization of synergistic hemorrhagins from the venom of the snake Bothrops asper

G. Borkow, J. M. Gutiérrez and M. Ovadia. Isolation and characterization of synergistic hemorrhagins from the venom of the snake Bothrops asper. Toxicon 31, 1137–1150, 1993.—Three hemorrhagic factors (BaH1, BH2 and BH3) were isolated from the venom of Bothrops asper by gel filtration on Sephacryl S-200, DEAE-Sepharose chromatography, metal chelate affinity chromatography and hydrophobic interaction chromatography. They contain 55% of the total hemorrhagic activity of the whole venom when they are mixed, but lose almost half of the activity if they are separated, indicating a synergism between the three. The main hemorrhagin is BaH1 ( Bothrops asper hemorrhagin 1); the other two are weak hemorrhagins but contribute to the synergism. They are acidic proteins with a p I of 4.5, 5.2 and 5; their mol. wt is 64,000, 26,000 and 55,000 respectively. The minimal hemorrhagic dose (MHD) of BaH1, BH2 and BH3 is 0.18, 2 and 1.6 μg, with a specific activity 55, 5 and 6.25 higher than that of the whole venom. The hemorrhagic activity of all three factors was inhibited by EDTA and ortho-phenathroline, indicating that the hemorrhagic activity is metal dependent. Phosphoramidon, soybean trypsin inhibitor, PMSF, pepstatin and aprotinin did not affect the hemorrhagic activity of the isolated factors.

Characterization of protein kinase C isotype expression in adult rat heart. Protein kinase C-epsilon is a major isotype present, and it is activated by phorbol esters, epinephrine, and endothelin.

The pattern of protein kinase C (PKC) isotype expression in whole extracts of dispersed, freshly isolated adult rat ventricular myocytes and adult rat heart ventricle was examined by immunoblot analysis using antisera specific for PKC-alpha, -beta 1, -gamma, -delta, -epsilon, -zeta, or -eta isotypes. This analysis revealed significant levels of expression of the Ca(2+)-independent isotype PKC-epsilon, which was detected as band of 97-kd molecular mass. PKC-zeta was detected principally as a 66-kd band that probably represented a proteolytic product of the holoenzyme. PKC-eta was detected only in whole ventricle as a doublet at 75 and 81 kd and was therefore probably present in nonmyocytic cells. PKC-alpha, -beta 1, -gamma, and -delta could not be detected. Because of our inability to detect PKC-alpha, -beta 1, -gamma, and -delta in whole extracts, PKC isotypes were partially purified from whole heart by DEAE Sepharose chromatography. PKC-alpha, -beta 1, -gamma, and -delta could still not be detected in the appropriate fractions. All PKC isotypes were detectable in appropriate positive control extracts (brain or certain cultured cell lines). In unstimulated isolated cardiomyocytes, the majority (80-95%) of the PKC-epsilon immunoreactivity was present in the soluble fraction of the extract. On exposure of the cardiomyocytes to 1 microM phorbol 12-myristate 13-acetate (PMA), PKC-epsilon undergoes a rapid (< 30 seconds), sustained (at least 60 minutes), and virtually complete association with the Triton X-100-soluble membrane fraction. There was an associated loss of PKC-epsilon from the soluble fraction. The EC50 for PMA of the translocation event was 15-37 nM. Exposure of cardiomyocytes to 1 microM 4 beta-phorbol 12,13-didecanoate or 1 microM phorbol 12,13-dibutyrate also resulted in translocation of PKC-epsilon to the membrane fraction, whereas exposure to 1 microM 4 alpha-phorbol 12,13-didecanoate was without effect. PKC-epsilon also translocated on exposure of cardiomyocytes to 50 microM epinephrine or 100 nM endothelin-1. However, in both cases, the extent of translocation was significantly less than that after exposure to PMA. We conclude that interventions that lead to hypertrophy of cardiomyocytes (phorbol esters, epinephrine, and endothelin-1) activate PKC-epsilon.

INTERACTION OF THE CYCLOPHOSPHAMIDE METABOLITE, ACROLEIN, WITH LACTATE DEHYDROGENASE-M HOMOLOG

The cyclophosphamide metabolite, acrolein, binds to both nucleic acids and proteins. In this report, acrolein is shown to interact preferentially with a rat liver 35KD DNA-binding protein. The 35KD protein was purified to homogeneity from a hepatic microsomal fraction using (NH4)2SO4 precipitation, DEAE-Sepharose chromatography, cation exchange HPLC, and DNA-Sepharose affinity chromatography. The protein was identified as a homologue of lactate dehydrogenase (LDH) M subunit on the basis of partial amino acid sequencing, amino acid composition and immunological methods. The interaction of this protein with acrolein may have implications for tissue-specific toxicities and/or oncoselectivity of cyclophosphamide.