Presence Of 2-mercaptoethanol Research Articles

The simultaneous determination of muramic acid and glucosamine in soil by high-performance liquid chromatography with precolumn fluorescence derivatization

The optimal release and quantitative estimation of muramic acid and glucosamine were studied simultaneously in soil samples. The effect of hydrolysis conditions, HCl concentration, hydrolysis time, the ratio of soil dry weight to acid, and the recovery of reference substances were investigated. Derivatization of the fluorogenic reagent o-phthalaldehyde, in the presence of 2-mercaptoethanol with the residue of a soil hydrolysate, was achieved by optimizing the relative amounts of o-phthalaldehyde to hydrolysate in the reaction mixture, the pH of both, and the incubation period. A linear relationship was found between the fluorescence response and the concentration of the test substances. The muramic acid, as well as the glucosamine (o-phthalaldehyde) derivatives gave single peaks, and complete separation from interfering substances at the picomol level was achieved in a short time (3 h preparation and 30 min for chromatography) by using high-performance liquid chromatography.

Determination of ammonium ion by fluorometry or spectrophotometry after on-line derivatization with o-phthalaldehyde

A fast, sensitive, simple, and highly reproducible method for routine assay of ammonium ion (NH4+) was developed by using HPLC equipment. The method is based on the reaction of NH4+ with o-phthalaldehyde (OPA) in the presence of 2-mercaptoethanol. After an on-line derivatization, the resulting NH4(+)-OPA product was quantified by using fluorometric or spectrophotometric detection. For fluorometric detection, the excitation and emission wavelengths were 410 and 470 nm, respectively. The spectrophotometric detection was made by measuring absorbance at 410 nm. Results on the effects of OPA-reagent composition and pH, reaction temperature, sample matrix, and linearity of the assay are presented. Even though it took about 2 min from the time of sample injection to the appearance of sample peak, sample injections could be overlapped at an interval of about 1 min. Thus, the actual time needed for analysis was about 1 min per assay. The method can be used in a fully automated mode by using an autosampler injector.

A new method for fluorometric measurement of proline in grapes and wines

A method for the measurement of free proline in grapes and wines containing other amino acids is presented. The method included the removal of phenols and tryptophan by the pre-treatment of samples with activated carbon, the oxidation of proline to the corresponding primary amine with sodium hypochlorite, destruction of other amino acids by oxidation at pH 5.3 using a combination of hydrogen peroxide, ferric chloride, and sodium hypochlorite, and the formation of afluorescent product from the oxidized proline with o-phthalaldehyde in the presence of 2-mercaptoethanol and Brij 35 at pH 5.3. The proline in grapes and wines measured by this method agreed well with the values obtained by the method using an automatic amino acid analyzer with ninhydrin.

Isoelectric focusing of basic proteins: the problem of oxidation of cysteines.

Isoelectric focusing of human globin chains in polyacrylamide gels dried in the ambient atmosphere and rehydrated in the presence of 8 mol/L urea produces artefactual doublets of zones as a result of oxidation by the gel. This oxidation can be avoided in separations of short duration by adding a reducing agent (e.g. 2-mercaptoethanol or dithiothreitol to the rehydration solution (Altland, K. and Rossmann, U., Electrophoresis 1985, 6, 314-325). We now demonstrate that the observed zone doublets can be explained by assuming neutralization of the contribution of dissociated sulfhydryl group of cysteine to pI by partial and reversible formation of globin dimers held together by disulfide bridges. Long time separations, requiring e.g. more than 4 h at greater than or equal to 500 V/cm, in pH gradients exceeding pH 7.5, are accompanied by artefactual oxidation from both the atmosphere and the gel matrix. Oxidation from the atmosphere as well as the effect of carbon dioxide can be eliminated by overlayering the gel with paraffin oil. Oxidation from the gel matrix can only partially be inhibited by rehydration of gels in the presence of 2-mercaptoethanol or dithiothreitol. Nearly complete protection against oxidation by the gel matrix was achieved by adding a permanent supply of 2-ME to the gel or by adding DTT to the cathodic wick towards the end of the experiment. Alkylation with iodoacetamide or iodoacetic acid resulted in stable globin patterns, which, however, displayed additional artefactual zones. Our experimental data indicate that the polyacrylamide gels function as an electron acceptor for dissociated sulfhydryl groups in proteins, even after pretreatment with strong reducing agents for proteins.

Purification and some properties of aminopeptidase from carp Cyprinus carpio ordinary muscle.

An aminopeptidase from carp Cyprinus carpio ordinary muscle was purified 700-fold by ammonium sulfate fractionation, ion-exchange chromatography, gel filtration and hydroxyapatite chromatography. The final enzyme preparation showed a single protein band on polyacrylamide gel electrophoresis (PAGE) and on sodium dodecyl sulfate (SDS) PAGE in the presence of 2-mercaptoethanol. The molecular weight of the enzyme was about 84, 000 and 94, 000 by gel filtration and SDS-PAGE, respectively. The enzyme was inhibited by p-chloromercuribenzoate, 5, 5'-dithiobis(2-nitrobenzoic acid), N-ethylmaleimide, p-tosyl-L-phenylalanine chloromethylketone, ptosyl-L-lysine chloromethylketone, EDTA, o-phenanthroline, puromycin and some heavy metal ions such as Cu2+, Zn2+, Hg2+ and Cd2+. The enzyme was activated by sulfhydryl compounds and Co2+. The enzyme had a broad specificity for hydrolysis of aminoacyl-β-naphthylamides (βNA), and pH optimum was 7.5. Ala-, Phe-, Lys-, Arg-, Leu-, Tyr- and Trp-βNA were suitable substrate for the enzyme, and Ser-, Pro-, Gly-, Val-, Ile- and Asp-βNA were poor substrate. Among the peptides used (Ala)5 and (Ala), were most rapidly hydrolyzed, and the relative rates of hydrolysis for both substrates were 2-fold higher than that of (Ala)3; and (Ala)3 was hydrolyzed about 60-fold higher than that of (Ala)2.

Purification and properties of an aminopeptidase from mullet, Mugil cephalus, Roe.

An aminopeptidase was purified from an aqueous extract of mullet roe in the presence of 2-mercaptoethanol by fractionation with ammonium sulfate and column chromatography on DEAE-cellulose and Sephadex G-200. The molecular weight of the enzyme was 184, 000 by gel filtration, and the enzyme appeared to consist of two homogenous subunits. The optimal pH and optimal temperature for activity were 7.4 and 45°C, respectively. Puromycin, p-chloromercuribenzoic acid, and o-phenanthroline inhibited the enzyme n on-competitively (their Ki=1.34μM, 0.113mM and 0.145mM, respectively), while 2-mercaptoethylamine was competitive (Ki = 0.056 mM). The enzyme was also inhibited by L-amino acids, in particular glutamic acid. The enzyme could hydrolyze a variety of α-aminoacyl β-naphthylamides and was most active on L-alanyl-β-naphthylamide. Judging from these properties, the mullet roe aminopeptidase resembles soluble alanyl aminopeptidase [EC 3.4.11.14].

Effect of 2-mercaptoethanol on the electrophoretic behavior of rat and dogfish metallothionein and chromatographic evidence of a naturally occurring metallothionein polymerization

1. Metallothionein behavior in SDS-PAGE has been characterized. 2. It has been found that metallothionein behavior in this electrophoretic system depends upon the reducing environment. Migration as a well-defined protein band is only achieved in the presence of 2–100 mM 2-mercaptoethanol. 3. Within those 2-mercaptoethanol levels, both rat and dogfish metallothionein migrate as a protein with a molecular weight several times higher than that expected by amino acid analyses. This is not due to molecule oxidations, since this effect is promoted by the presence of 2-mercaptoethanol. 4. No effect of 2-mercaptoethanol on metallothionein behavior is found in conventional PAGE. 5. The present results suggest that to study the effect of 2-mercaptoethanol in SDS-PAGE is a simple and accurate way to identify a protein as metallothionein. 6. It has also been found that metallothionein aggregates naturally in the absence of ionic strength.

Some problems arising in SDS-PAGE and silver staining of reduced protein samples

The presence of 2-mercaptoethanol or dithiothreitol in the protein denaturing solution used to prepare samples for SDS-PAGE gives rise to artifacts by silver staining of the gel. Addition of iodoacetamide to the reduced protein sample prevents the appearance of artifacts. Silver staining is strengthened by the dithiothreitol treatment but weakened by subsequent treatment with iodoacetamide. High concentration of dithiothreitol also inhibited development of silver staining. These effects of iodoacetamide or high concentration of dithiothreitol were observed more pronouncedly with the silver-staining method of Merril et al.4) than of Guevara.5) Mobility of the protein having intramolecular disulfide bonds is more or less slowed down depending on the concentration of dithiothreitol used. From these results it is concluded that the most suitable concentration of dithiothreitol is in the range of 40 to 80mM from the viewpoints of sensitivity of silver staining, completion of mobility shift, and sharpness of developed protein bands.

Isolation and purification of a rat liver-specific antigen from hepatocyte membrane.

A rat liver-specific antigen (RLSA) solubilized with the nonionic detergent non-anonyl-N-methylglucamide was purified through affinity column chromatography with a monoclonal antibody and by high-performance liquid chromatography with a hydroxylapatite column. The purified RLSA showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its molecular weight was determined to be 105,000 in the presence of 2-mercaptoethanol. The antigen was reactive to the Schiff reagent and contained glucosamine, but not galactosamine, indicating that the RLSA is a glycoprotein containing an asparagine-binding type of sugar chain.

Purification and in vitro complementation of mutant histidinol dehydrogenases.

The biochemistry of interallelic complementation within the Salmonella typhimurium hisD gene was investigated by in vitro protein complementation of mutant histidinol dehydrogenases (EC 1.1.1.23). Double-mutant strains were constructed containing the hisO1242 (constitutive overproducer) attenuator mutation and selected hisDa or hisDb mutations. Extracts from such hisDa986 and hisDb1799 mutant cells failed to show histidinol dehydrogenase activity but complemented to produce active enzyme. Inactive mutant histidinol dehydrogenases were purified from each of the two mutants by ion-exchange chromatography. Complementation by the purified mutant proteins required the presence of 2-mercaptoethanol and MnCl2, and protein-protein titrations indicated that heterodimers were strongly preferred in mixtures of the complementary mutant enzymes. Neither mutant protein showed negative complementation with wild-type enzyme. The Vmax for hybrid histidinol dehydrogenase was 11% of that for native enzyme, with only minor changes in Km values for substrate or coenzyme. Both purified mutant proteins failed to catalyze NAD-NADH exchange reactions reflective of the first catalytic step of the two-step reaction. The inactive enzymes bound 54Mn2+ weakly or not at all in the presence of 2-mercaptoethanol, in contrast to wild-type enzyme which bound 54Mn2+ to 0.6 sites per monomer under the same conditions. The mutant proteins, like wild-type histidinol dehydrogenase, behaved as dimers on analytical gel filtration chromatography, but dissociated to form monomers in the presence of 2-mercaptoethanol. This effect of 2-mercaptoethanol was prevented by low levels of MnCl2. It thus appears that mutant histidinol dehydrogenase molecules bind metal ion poorly. The complementation procedure may allow for formation of a functional Mn2+-binding site, perhaps at the subunit interface.

Protein phosphatases of the guinea-pig parotid gland.

The nature of protein phosphatases of the guinea-pig parotid gland was investigated. The protein phosphatases were characterized by (a) the use of five different 32P-labelled substrate proteins (phosphorylase a, histone H2B, casein, and the alpha and beta subunits of phosphorylase kinase), (b) their behaviour during ion-exchange chromatography, (c) their relative molecular mass distribution during gel filtration, (d) their sensitivity towards inhibition by inhibitor 2, (e) their ability to be stimulated by protamine and (f) by their behaviour during freezing and thawing in the presence of 2-mercaptoethanol. The following results were obtained. 1. The 'cytosol' (100,000 X g supernatant) contains protein phosphatases of the types 1, 2A and 2B. 2. On the basis of inhibition with inhibitor 2 (1.2 micrograms/ml) the 'cytosolic' phosphorylase phosphatase activity consists to about 40% of protein phosphatase 1 and to about 60% of protein phosphatase 2A. 3. In the cytosol about 80-90% of the protein phosphatases 1 and 2A exist in an inactive state. 4. A 5-10-fold activation can be achieved by ethanol precipitation, which results in the generation of a mixture of forms of low apparent molecular mass of about 30 kDa. 5. Microsome-associated phosphorylase phosphatase activities can be extracted in a highly active state by detergent (1% Triton X-100) or by 0.8 M NaCl. 6. Activity measurements in the presence of inhibitor 2 (1.2 micrograms/ml) indicate that the microsomal activities consist to about 75% of protein phosphatase 1 and to about 25% of protein phosphatase 2A. Activities corresponding to protein phosphatases 2B and 2C could not be detected. 7. The 'microsomal' protein phosphatase activities exhibit lower apparent molecular masses (70 kDa and 30 kDa) than the 'cytosolic' protein phosphatases (about 260 kDa). 8. After ethanol treatment of the microsomal protein phosphatases only activities with apparent molecular masses of about 30 kDa can be detected. These share several similarities with the ethanol-treated cytosolic protein phosphatases. 9. Both cytosolic and microsomal protein phosphatases display activity towards histone H2B and casein.

The role of benzodiazepine receptors in the induction of differentiation of HL‐60 leukemia cells by benzodiazepines and purines

A series of benzodiazepines was evaluated for their capacity to induce the differentiation of HL-60 acute promyelocytic leukemia cells. Benzodiazepines were effective initiators of maturation in the concentration range of 50 to 150 microM. The possible involvement of benzodiazepine receptors in mediating the differentiation induced by these agents was investigated. The presence of high affinity, peripheral type benzodiazepine binding sites (KD = 7.3 nM, TB = 14.5 pmol/mg protein with Ro5-4864) was demonstrated in HL-60 membranes. The occupancy of peripheral type high affinity benzodiazepine receptors by various benzodiazepines showed some correlation (r = 0.76) with their differentiation-inducing capabilities, but binding potencies were 1,000-fold higher than the concentrations required to produce differentiation. A class of benzodiazepine receptors with lower binding affinity was also detected in HL-60 membranes (KD = 28.6 microM; TB = 199 pmol/mg protein with diazepam). A higher level of correlation (r = 0.88) was demonstrated between benzodiazepine occupancy of these lower affinity receptors and the capacity to induce maturation. Significantly, benzodiazepine concentrations needed for low affinity binding and induction of differentiation were the same (25-200 microM), suggesting that low affinity benzodiazepine receptors may be involved in the induction process. We have shown that the molecular form responsible for the induction of the differentiation of HL-60 cells to mature forms by 6-thioguanine (TGua) is the free base, TGua, itself [Ishiguro, Schwartz, and Sartorelli (1984) J. Cell. Physiol., 121:383-390]. Since hypoxanthine (Hyp) and inosine (Ino) have been identified as putative endogenous ligands for high affinity benzodiazepine receptors in brain tissue, the potential involvement of benzodiazepine receptors in the differentiation of HL-60 cells by the purines was investigated. Physiological purines such as Hyp and Ino were inactive in displacing the benzodiazepines from their high and low affinity binding sites in HL-60 membranes. In contrast, TGua caused inhibition of benzodiazepine binding to high and low affinity sites. The inhibition of Ro5-4864 binding to high affinity binding sites by TGua appeared to be due to the binding of TGua to membranes through the formation of a mixed disulfide between the 6-thiopurine and protein thiols, since the inhibition was reversed by the presence of 2-mercaptoethanol. The findings suggest a possible relationship between the occupancy of benzodiazepine receptors by TGua and the induction of leukemic cell differentiation.

Interaction of a lymphokine with normal human macrophages results in release of a suppressor factor for mitogen-induced immunoglobulin synthesis.

Normal human macrophage/monocyte cultures exposed to a suppressor factor produced by concanavalin A-activated T cells (T-SF), respond by releasing after 72 h a macrophage-derived suppressor factor (M phi-SF). The M phi-SF inhibits pokeweed mitogen-induced Ig synthesis but not T- or B-cell proliferation. Cycloheximide treatment of the macrophages does not interfere with generation of the M phi-SF, suggesting that de novo synthesis is not required. The factor is not preformed, for virgin macrophages do not contain M phi-SF, but it appears in macrophage cell lysates after exposure to T-SF. The production of the M phi-SF is inhibited by the presence of 2-mercaptoethanol. Both T-SF and M phi-SF are L-rhamnose inhibitable, and the M phi-SF appears to be released as a high molecular weight complex which is dissociable into a low molecular weight form of a size similar to the T-SF, i.e. approximately 20,000. The T-SF induced M phi-SF has some similarities with soluble immune response suppressor (SIRS) but differs from this factor in its lack of effect upon lymphocyte proliferation failure to induce conversion of T-SF to M phi-SF by treatment with H2O2.

Selective labeling of sulfhydryls and disulfides on blot transfers using avidin-biotin technology: Studies on purified proteins and erythrocyte membranes

Various strategies for the use of 3-( N-maleimido-propionyl) biocytin (MPB) as a general label for distinguishing between protein sulfhydryls and disulfides on blot transfers are presented. In the first approach, endogenous SH groups in proteins were labeled directly with MPB. For disulfide staining, endogenous sulfhydryls were blocked with N-ethylmaleimide, disulfides were then reduced with mercaptoethanol, and the newly formed SH groups were labeled with MPB. In this approach, all treatments were performed in vitro, and, between steps, excess reagent was removed by dialysis. The MPB-labeled proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (in the presence of mercaptoethanol), the labeled proteins were transferred to nitrocellulose, and the blotted proteins were detected by avidin-biotin technology. In the second approach, MPB treatment was performed directly on blot transfers. For SH labeling, proteins were subjected to SDS-PAGE in the absence of mercaptoethanol, thus retaining the status of endogenous sulfhydryl and disulfide groups. For SS labeling, proteins were treated with N-ethylmaleimide in vitro and then subjected to SDS-PAGE in the presence of mercaptoethanol, such that endogenous sulfhydryls were blocked and endogenous disulfides were converted to SH groups. Subsequent treatments and washings were performed on blots. In the third approach, immobilized proteins (i.e., in artificial systems or in natural systems such as membrane preparations or intact cells) were treated essentially as described in the first approach, except that washings were carried out by centrifugation. In vitro treatments were performed before SDS-PAGE (carried out in the presence of mercaptoethanol) and subsequent blot transfer. The relative merits of the three strategies are discussed.

Simultaneous determination of histidine and histamine by second-derivative synchronous fluorescence spectrometry

Simultaneous direct determination of histidine and histamine in mixtures is described. The method is based on the formation of fluorescent condensation products with o-phthaldialdehyde in a basic medium in the presence of 2-mercaptoethanol. Both the conventional and synchronous fluorescence spectra of these condensation products completely overlap, making determination by either of these techniques impossible. However, the determination can be performed by second-derivative synchronous fluorescence spectrometry. The method is simple, rapid and inexpensive and the measurements are performed in a single scan. Calibration graphs are linear in the range 0.1–4.0 μg/ml for histidine and 0.06–5.0 μg/ml for histamine. Mixtures of histidine and histamine in ratios between 12:1 and 1:8 have been satisfactorily resolved.

Control of nitrogen assimilation in Stichococcus bacillaris by growth conditions

Stichococcus bacillaris Naeg. (Chlorophyceae) grown on a 12 h light: 12 h dark cycle divides synchronously under photoautotrophic conditions and essentially nonsynchronously under mixotrophic conditions. Photoassimilation of carbon under photoautotrophic conditions was followed by a decline in cell carbon content during the dark period, whereas under mixotrophic conditions cell carbon increased throughout the light–dark cycle. The rates of nitrogen assimilation by cultures grown on either nitrate or ammonium declined sharply during the dark, and these declines were most pronounced under photoautotrophic conditions. Photoautotrophic cells synthesized glutamine synthetase and NADPH – glutamate dehydrogenase (GDH) exclusively in the light, whereas in mixotrophic cells about 20% of the total synthesis of these enzymes during one light–dark cycle occurred in the dark. NADH–GDH was synthesized almost continuously over the entire light–dark cycle. In the dark, both under photoautotrophic and mixotrophic conditions, the alga contained more than 50% of glutamine synthetase in an inactive form, which was reactivated in vitro in the presence of mercaptoethanol and in vivo after returning the cultures to the light. The thermal stability of glutamine synthetase activity was less in light-harvested cells than in dark-harvested cells. The inactivation of glutamine synthetase did not occur in cultures growing either heterotrophically in continuous darkness or photoautotrophically in continuous light. This enzyme appears to be under thiol control only in cells grown under alternating light–dark conditions, irrespective of whether this light regime results in synchronous cell division or not.

Isolation and characterization of agglutinins from the hemolymph of an acorn barnacle, [formula omitted][formula omitted

Two agglutinins. MVA-1 and MVA-2, were isolated from the hemolymph of the acorn barnacle, Megabalanus volcano . They agglutinated human erythrocytes irrespective of the ABO blood group and also rabbit and sheep blood cells. Lactose and fetuin strongly inhibited the hemagglutinating activity. D-galactose, D-arabinose and N-acetylneuraminic acid were also moderate inhibitors. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both MVA-1 and MVA-2 gave a single band corresponding to 38,000 daltons. It split into one major band with a molecular weight of 23,000 in the presence of 2-mercaptoethanol. The two agglutinins showed the same apparent molecular weight of 116,000 by gel filtration. In isoelectric focusing MVA-1 showed one band at pH 4.8. whereas MVA-2 gave a main band at pH 4.4 with few faint ones in the range between pH 4.0 and 4.8. The agglutinins were glycoproteins containing D-mannose and L-fucose as carbohydrate components. No precipitation reaction was observed in Ouchterlony immuno-diffusion tests using rabbit antisera against the agglutinins from the phylogenetically related Megabalanus rosa .

Purification and characterization of human colony-stimulating factor 1 from human pancreatic carcinoma (MIA PaCa-2) cells

Colony-stimulating factor 1 (CSF-1) was purified from the serum-free conditioned medium of a human pancreatic carcinoma cell line (MIA PaCa-2) by a combination of conventional chromatography and high-performance liquid chromatography. The purity of human CSF-1 was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a diffuse single band of M r 42,000–50,000 and by N-terminal amino acid analysis of glutamate residue. The CSF-1 was stable at 50 °C for 30 min. It is sensitive to treatment with trypsin, chymotrypsin, and subtilisin but less sensitive to papain digestion. Treatment of CSF-1 with different glycosidases did not affect the biological activity. Sulfhydryl reagents such as dithiothreitol (DTT), iodoacetic acid, and N-ethylmaleimide did not affect the biological activity at the concentration of 1 m m. However, CSF-1 activity was inhibited totally by the combination of 10 m m DTT and 1 m m SDS. Under denaturing and reducing conditions, CSF-1 appeared on SDS-PAGE as a single protein band of M r 21,000–25,000 and concurrently lost its activity, indicating that human CSF-1 possibly consists of two similar subunits and that the intact quaternary structure is essential for the biological activity. When treated with neuraminidase and endo-β- d- N-acetylglucosaminidase D, the molecular weight of CSF-1 was reduced to 36,000–40,000, and to 18,000–20,000 in the presence of mercaptoethanol. Because of the specificity of endo-β- d- N-acetylglucosaminidase D, it is suggested that the carbohydrate moieties are Asn-linked “complex-type” units.

Purification and characterization of the dihydropyridine-sensitive voltage-dependent calcium channel from cardiac tissue.

The dihydropyridine-sensitive voltage-dependent Ca2+ channel from cardiac tissue was purified 900-fold using DEAE-Sephadex A-25, concanavalin A-Sepharose, and wheat germ agglutinin-Sepharose. The purified preparation was highly enriched in a peptide of 140,000 daltons when electrophoresed on sodium dodecyl sulfate gels in the presence of 2-mercaptoethanol, or 170,000 when electrophoresed in the presence of iodoacetamide. Polyclonal antibodies raised against the purified subunits of the rabbit skeletal muscle Ca2+ channel recognized the 170-kDa protein in preparations electrophoresed under nonreducing conditions, and the large peptide of 140 kDa and smaller peptides of 29-32 kDa in preparations analyzed under reducing conditions. Monoclonal antibodies, which were raised against the native Ca2+ channel from skeletal muscle, immunoprecipitated [3H]PN 200-110 binding activity from solubilized cardiac membranes and immunoprecipitated 125I-labeled peptides (from the purified cardiac Ca2+ channel preparation) which migrated as a single species of 170 kDa under nonreducing conditions, or as 140, 32, and 29 kDa under reducing conditions. The results show that the purified cardiac Ca2+ channel, like that previously purified from skeletal muscle, consists of a major component of 170 kDa which is comprised of a 140-kDa peptide linked by disulfide bonds to smaller peptides of 32-29 kDa. Peptide maps of the 140-kDa peptide purified from cardiac and skeletal muscle preparations were strikingly similar, suggesting a high degree of homology in their primary sequence.

Spectroscopic properties of spinach catalase

Spinach catalase (hydrogen-peroxide: hydrogen-peroxide oxidoreductase, EC 1.11.1.6) has been purified to homogeneity. The purified enzyme has a specific activity of 25 000 units per mg protein. The presence of 2-mercaptoethanol and phenylmethylsulfonyl fluoride (PMSF) were required for high yields of the enzyme. The molecular weight of the enzyme was estimated to be 125 000 by gel filtration. Subunit analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed a single peptide with M r 55 000. The enzyme, which exhibits optical absorbance maxima at 279, 403, 542, 592 and 723 nm and shoulders at 290, 500 and 630 nm, contains 2 mol iron per mol protein. One of the two irons can be attributed to protoheme, while the other iron appears to be present in a novel heme. The oxidized catalase exhibited two sets of high-spin, ferriheme EPR signals.