Isolation and partial characterization of an arginine-rich apolipoprotein from human plasma very-low-density lipoproteins: apolipoprotein E.

A water-insoluble apoprotein was isolated from apo-VLDL by column chromatography on Sephadex G-200 in sodium dodecylsulfate followed by preparative polyacrylamide gel electrophoresis in a discontinous sodium dodecylsulfate system, or by preparative electrophoresis alone. The protein was similar in amino acid composition to the "arginine-rich protein" reported by Shore and Shore. It represented about 10% of the total protein mass of VLDL. The apoprotein showed one single band with an apparent Mr of 39000 in sodium dodecylsulfate gel electrophoresis, and was homogeneous in gel electrophoresis at pH 8.9 In 8M urea. Immunochemical studies also showed homogeneity of this protein, and antisera prepared against it did not react with any other of the well known apolipoproteins, but did react with VLDL and apo-VLDL preparations. Analytical isoelectric focusing in 8M urea resulted in a heterogeneous banding pattern showing three major polypeptides with pI values of 5.5, 5.6 and 5.75. Thus this apolipoprotein clearly differs from the apo-B and apo-C polypeptides of VLDL as well as from apoproteins A and D in its molecular weight, amino acid composition, focusing behavior and immunochemical properties.     

Superoxide dismutase in the housefly,Musca domestica (L.)

Four superoxide dismutase (SOD) (E.C. 1.15.1.1) isozymes were present in whole tissue homogenates of Musca domestica when examined by polyacrylamide gel electrophoresis. One of the isozymes contained manganese, and the other three contained copper and zinc. All were observed in each of the body tagma (head, abdomen, and thorax) and at each developmental stage (egg to adult). The copper- and zinc-containing isozymes purified from newly emerged, adult M. domestica had a relative molecular weight of 34,800 as determined by gel filtration chromatography but consisted of two equal-size subunits of 16,000 as measured by sodium dodecylsulfate polyacrylamide gel electrophoresis. An isoelectric point between 4.8 and 5.1 was measured. Approximately 2 mol each of copper and zinc were present per dimer. The three copper, zinc isozymes were identified as charge variants. The amino acid composition of the enzyme was similar to that of copper, zinc-containing superoxide dismutases from other sources. Purified housefly copper, zinc superoxide dismutase was neither deactivated nor able to protect lactic dehydrogenase against deactivation in the presence of light and rose bengal, a known generator of singlet oxygen. The role of SOD in the phototoxic reaction involving rose bengal is discussed.     

Sugar transport by the bacterial phosphotransferase system. Characterization of the sulfhydryl groups and site-specific labeling of enzyme I

Enzyme I is the first protein of the phospho transfer sequence in the bacterial phosphoenolpyruvate:glycose phosphotransferase system. This protein exhibits a temperature-dependent monomer/dimer equilibrium. The nucleotide sequence of Escherichia coli ptsI indicates four -SH residues per subunit (Saffen, D. W., Presper, K. A., Doering, T. L., and Roseman, S. (1987) J. Biol. Chem. 262, 16241-16253). In the present experiments, the sulfhydryl groups of the E. coli enzyme were studied with various -SH-specific reagents. Titration of Enzyme I with 5,5'-dithiobis-2-nitrobenzoic acid also revealed four reacting -SH groups. The kinetics of the 5,5'-dithiobis-2-nitrobenzoic acid reaction with Enzyme I exhibit biphasic character, with pseudo-first order rate constants of 2.3 x 10(-2)/s and 2.3 x 10(-3)/s at pH 7.5, at room temperature. Fractional amplitudes associated with the rate constants were 25 +/- 5% for the fast and 75 +/- 5% for the slow rate. The "slow" rate was influenced by ligands that react with Enzyme I (the protein HPr, Mg2+, Mg2+ plus P-enolpyruvate), and also by temperature (at the temperature range where the monomer/dimer association occurs). The fractional ratio of the two rates remained at 1:3 under these conditions. Thus, under all conditions tested, two classes of -SH groups were detected, one reacting more rapidly than the other three -SH groups. Modification of the "fast" -SH group results in an active enzyme capable of forming dimer, whereas modification of the slow -SH groups results in inactive and monomeric Enzyme I. The enzyme was labeled with pyrene maleimide under conditions where only the more reactive sulfhydryl group was derivatized. Hydrolysis by trypsin followed by reverse-phase high performance liquid chromatography analysis of the peptide mixture resulted in only one fluorescent peak. This peak was not observed when the more reactive sulfhydryl residue was protected prior to pyrene maleimide labeling. Amino acid sequencing of the fluorescent peak indicated that the more reactive residue is the C-terminal amino acid residue, cysteine 575. The results provide a means for selectively labeling Enzyme I with a fluorophore at a single site while retaining full catalytic activity.     

Bovine kidney alkaline phosphatase. Purification, subunit structure, and metalloenzyme properties.

Kidney alkaline phosphatase was purified to homogeneity. It is a glycoprotein of about 172,000 molecular weight. Analyses of the subunit structure by sedimentation equilibrium in 6 M guanidine hydrochloride and by gel electrophoresis in sodium dodecyl sulfate indicate that the alkaline phosphatase is a dimer comprising two very similar or identical subunits of about 87,000 molecular weight. The native enzyme contains 4.5 +/- 0.2 g atoms of zinc per mol of protein. Reconstitution experiments from the apophosphatase show that binding of 4 Zn2+ per mol of dimer is essential for full activity. The kinetic data of Zn2+ binding to the apoprotein require at least a two-step mechanism, in which one of the steps corresponds to a conformational change within the enzyme. This paper also presents data concerning amino acid composition, sugar content, enzyme stability, absorbance index, and sedimentation velocity.     

Characterization and substrate specificity of fumarylacetoacetate fumarylhydrolase.

The molecular weight of fumarylacetoacetate fumarylhydrolase (EC 3.7.1.2) is 86 000 +/- 10 000, as determined by gel filtration. The enzyme appears to be a dimer with a monomer molecular weight of 38 000 - 43 000, as determined by gel electrophoresis, gel filtration in guanidine-hydrochloride, and ultracentrifugation. The subunits appear to be identical, as only one band is seen in gel electrophoresis, only one protein peak is detected in gel filtration in guanidine-hydrochloride, and only one amino-terminal amino acid (proline) is detected. Three free sulfhydryl groups per denatured monomer are detected by reaction with 5,5'-dithiobis(2-nitrobenzoic acid), while for the active enzyme only two sulfhydryl groups react with this reagent, The extinction coefficients at 260 and 280 nm, the amino acid composition, and the isoelectric point (6.7) of the enzyme are also reported. The enzyme catalyzes the hydrolysis of six 2,4-diketo acids and three 3,5-diketo acids tested. The Km of the substrates is similar but V varies by a factor of 120. The pH optimum is 7.3. The enzyme did not catalyze the hydrolysis of a number of esters tested.     

Protein components in the very low density lipoproteins of hen's egg yolks. Identification of highly aggregating (gelling) and less aggregating (non-gelling) proteins.

Apoproteins of hen's egg yolk very low density lipoprotein has been separated by Sephadex G-200 gel filtration in 0.5% sodium dodecyl sulfate into three categories of proteins termed apoprotein A, apoprotein B and apoprotein C. Apoprotein A fraction consists of several aggregated proteins (linked possibly by -S-S- bridges) as shown by acrylamide gel electrophoresis in the presence of 2-mercaptoethanol. Apoprotein B contains two major protein components, B1 and B2, with molecular weights of 78 000 and 64 000, respectively, and two minor proteins components. Apoprotein C was obtained in a pure form as a low molecular weight, -S-S- linked dimer protein and accounted for about 30% of the total protein. In the monomeric form, apoprotein C has a molecular weight of 9400. Apoprotein A and apoprotein B have similar amino acid composition, except in isoleucine content which is over two times in apoprotein B as compared to apoprotein A. Apoprotein C lacks histidine and is richer in arginine than apoproteins A or B. Apoprotein C has lysine as N-terminal, while apoproteins A and B have predominantly arginine as the N-terminal amino acid. All the three fractions contain carbohydrate residues, apoprotein B being the richest in carbohydrate content. Cold-stored apoproteins A forms a clear gel when dispersed in 0.5% sodium dodecyl sulfate at concentration of above 2 mg/ml, while apoprotein B forms a gel only above 10 mg/ml. Apoprotein C, even at 35 mg/ml, forms a clear solution with no tendency to gel.     

Purification of a non-chloroplastic α-glucan phosphorylase from spinach leaves

The non-chloroplastic -glucan phosphorylase (EC 2.4.1.1) from spinach leaves has been purified to homogeneity as revealed by dodecylsulfate gel electrophoresis. Both purification and separation from the chloroplastic phosphorylase were achieved by chromatography on Sepharose-bound dextrin. The chloroplastic phosphorylase did not bind to Sepharose-dextrin and was removed from the column by washing with buffer, as verified by polyacrylamide gel electrophoresis of the buffer eluate and by chromatography of a preparation from isolated intact chloroplasts. The non-chloroplastic phosphorylase did bind to a high extent to Sepharose-dextrin and could be eluted by a dextrin gradient. Based on dodecylsulfate gel electrophoresis and pyridoxal phosphate determination, a molecular weight of about 90,000 was found for the monomer. Molecular-weight determination by porosity density gradient electrophoresis and gel filtration on Sephadex G-200 suggested that the native enzyme is a dimer, as are other phosphorylases.Abbreviations DEAE diethylaminoethyl - EDTA ethylenediamine tetraacetic acid - G1P glucose 1-phosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - PMSF phenylmethyl sulphonyl fluoride - SDS sodium dodecylsulfate - Tris Tris (hydroxymethyl)aminomethane Dedicated to Professor Dr. A. Pirson on the occasion of his 70th birthday     

Apolipoprotein A-IV: a protein occurring in human mesenteric lymph chylomicrons and free in plasma. Isolation and quantification.

1. Human mesenteric lymph chylomicrons were isolated from chylous ascites fluid by ultra-centrifugation and agarose/gel chromatography and their apoprotein composition was analysed by dodecylsulfate/polyacrylamide gel electrophoresis, analytical isoelectric focusing and immuno-chemically. Major components of mesenteric lymph chylomicrons were apoprotein A-I, proteins of Mr less than 15 000 including the C-group apoproteins and a protein of Mr 46 000. Minor components were apoprotein E and a protein of Mr approximately equal to 200 000 (B-like protein). This apoprotein composition was qualitatively identical with that of chylomicrons from intestinal lymph of the rat, but was distinctly different from plasma chylomicrons of humans with fasting chylomicronaemia. 2. The protein of Mr approximately equal to 46 000 has been isolated by preparative dodecylsulfate/polyacrylamide gel electrophoresis from human and rat lymph chylomicrons and was compared to a protein of identical Mr present in rat high-density lipoproteins (apoplipoprotein A-IV) and in the rho less than 1.006 g/ml serum lipoprotein fraction of individual humans with alimentary hypertriglyceridaemia. In both species the 46 000-Mr proteins isolated from lymph and serum were identical according to amino acid composition and isoelectric point in 6 M urea. The human proteins from both sources were also immunologically identical. The similarities in the molecular properties of the human apolipoprotein and rat apolipoprotein A-IV indicate that these proteins are homologous. 3. Plasma levels of human apolipoprotein A-IV determined by electroimmunodiffusion were 14.15 +/- 3.66 mg/100 ml (n = 59), but greater than 90% of the protein was unassociated with the major lipoprotein fractions. It is concluded, that apolipoprotein A-IV is a main protein component of human lymph chylomicrons, that is removed from the particles in the plasma compartment.     

Chemical investigations on pig kidney aminoacylase.

1. Preparations of purified pig kidney aminoacylase (N-Acylamino-acid amidohydrolase, EC 3.5.1.14) were obtained by Sephadex and DEAE-cellulose chromatography in homogeneous form as judged by polyacrylamide gel electrophoresis and immunoelectrophoresis. 2. The apparent molecular weight of the enzyme, determined by gel filtration, was about 86 000. After treatment with mercaptoethanol, performic acid or sodium dodecyl sulphate a band with an apparent molecular weight of approximately 43 000 was observed in polyacrylamide gels containing sodium dodecyl sulphate. Thus pig kidney aminoacylase seems to be composed of two subunits. 3. The amino acid composition of the enzyme was determined. Aminoacylase contains 772 amino acids, which corresponds to a molecular weight of 85 500. 12 tryptophan and 12 half-cystine residues were found. 4. Each subunit of the enzyme contains two -SH groups of different reactivity and two disulfide bonds one of which is easily cleaved by -SH compounds, the second only by performic acid oxidation. 5. Chemical modification of two -SH groups abolishes the catalytic activity of aminoacylase. Cleavage of two disulfide bonds also inactivates the enzyme. It is suggested that the enzyme has two active sites each containing an essential -SH group and disulfide bond. One active site is assumed to be part of each subunit.     

Hemocyanins in spiders, IV[1]. Subunit heterogeneity of Eurypelma (Dugesiella) hemocyanin, and separation of polypeptide chains.

The hemocyanin of the North American tarantula Eurypelma californicum (Dugesiella californica) is dissociated at pH 9.6 into monomers (Mr about 70 000) and dimers (Mr about 140 000), which were separated by gel filtration. The monomer peak was resolved by preparative polyacrylamide gel electrophoresis and yielded 4 protein bands, three of which (1, 3 and 4M) are apparently homogeneous. Band 2 contains two sub-fractions (2I and 2II). The dimer peak contains two dimers (bands 4D and 5). Upon treatment with 5mM cysteine the dimer band 5 is dissociated, yielding only one type of monomer identical with band 3. The other dimer, which was only partially dissociated by 10mM EDTA, is most probably a heterodimer, one component being electrophoretically indistinguishable from band 2II. After treatment of the native hemocyanin with sodium dodecylsulfate and analysis in gradient gel slabs, 6 polypeptide chains were observed (labeled a - f). They correspond to the products of alkaline dissociation as follows: band 1 = e, band 2I = a, band 2II = c, band 3 = f, band 4M = d, band 4D = b plus c, band 5 = f. The molecular weights were determined by dodecylsulfate gel electrophoresis in gradient gels, and by sedimentation equilibrium analysis and found to range between 67 000 and 76 000. The sedimentation coefficients are between 4.4 and 4.7 S for the monomers and 6.6 and 6.7 for the dimers. The isoelectric points range from pH 4.5 to pH 5.4. The findings are discussed with respect to the limitations of molecular weight determination by conventional dodecylsulfate gel electrophoresis, to the structure of the hemocyanin oligomers and to possible biological significance.     

Subunit structure and activity of glyceraldehyde-3-phosphate dehydrogenase from spinach chloroplasts.

Glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate : NADP+ oxidoreductase (phosphorylating), EC 1.2.1.13) from spinach chloroplasts is a polymeric protein of approx. 600,000 daltons and sodium dodecyl sulphate gel electrophoresis shows that it consists of two subunits of molecular weight 43,000 and 37,000. Comparison of amino acid analyses and tryptic peptide maps indicates that the two subunits have a different primary structure. The native enzyme contains 0.5 mol of NADP+ and 0.5 mol of NAD+ per protomer of 80,000 daltons, no reduced pyridine nucleotides have been detected. Almost complete inactivation is obtained by reaction of two cysteinyl residues per 80,000 daltons with tetrathionate or iodo[14C2]acetic acid; since the same amount of radioactivity is incorporated in the two subunits it is likely that they are both essential for the catalytic activity. Charcoal stripping of native glyceraldehyde-phosphate dehydrogenase produces an apoprotein which still retains most of the enzymatic activity but, unlike the holoenzyme, is gradually inactivated by storage at 4 degrees C and does not react with iodoacetate under the same conditions in which the holoenzyme is completely inactivated.     

The reactivity of -SH groups in the ADP/ATP carrier isolated from beef heart mitochondria

The emergence of the reactivity of -SH groups associated with conformation changes has been studied on the ADP/ATP carrier, is isolated in three different inhibitor-protein complexes. 1. The bongkrekate-protein complex incorporates approximately one molecular more of N-ethylmaleimide than the carboxyatractylate-protein complex. After extensive denaturation by dodecylsulfate in urea, both inhibitor complexes exhibit four reactive -SH groups per subunit. Thus one of four -SH groups per subunit has been unmasked in the bongkrekate-protein complex. 2. The interconversion from the bongkrekate-protein complex to the carboxyatractylate-protein complex is inhibited after the -SH groups have been blocked. 3. The protein complex isolated with the more easily dissociable atractylate, is used to demonstrate, by the emergence of the -SH groups, the transition into the m-state. This transition is specifically catalyzed by ADP and ATP. 4. Using 2,2'-dinitro-5,5'-dithiodibenzoate, the appearance of the -SH groups on transition from the c-state to the m-state can be followed spectrophotometrically. The specificity for the catalyzing nucleotides is identical with that for the transport. The Km for ADP and ATP is in the range of 1 microM. In conclusion, the thiol groups of the isolated ADP/ATP carrier behave as in the mitochondrial membrane. The unmasking of -SH groups is in full accordance with the concept of two conformational states (c and m).     

A type kappa Bence Jones protein containing a cysteinyl residue in the variable region.

The proteins precipitated with ammonium sulfate from the urine of a patient (Mat) with multiple myeloma were separated into three components by ion-exchange and gel chromatographies. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, amino acid analyses, immunochemical tests, and measurement of circular dichroism showed that these components were a dimer with a disulfide bond, a stable monomer, and a variable fragment, respectively. All three protein components reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) in Tris-HCl buffer at pH 8.0, indicating that they contained free sulhydryl groups. Partial reduction with dithiothreitol in the absence of denaturants yielded two SH groups per molecule from both the monomer and the dimer, and one SH group per molecule from the fragment. This indicates that the monomer of Mat protein contains a cysteinyl residue in the variable region in addition to a cysteinyl residue at the COOH terminus.The reactivities of the two SH groups of the partially reduced monomer toward iodoacetamide and iodoacetic acid were studied by polyacrylamide gel electrophoresis. The two SH groups had similar reactivities with iodacetamide, but the SH group at the COOH terminus was more reactive with iodoacetic acid than that in the variable region. The extrinsic Cotton effects of an azobenzene-2-sulfenyl group introduced into the SH group in the variable region were different from those of dye attached to the COOH terminal SH group, indicating that the two SH groups had different environments. The states of the SH groups of the intact monomer are discussed on the basis of these findings.     

Characterization of a new murein-associated lipoprotein in the outer membrane of Proteus mirabilis

A murein-associated outer membrane protein from Proteus mirabilis has been isolated. Since the protein carries ester- as well as amide-linked fatty acids it can be classified as a second outer membrane lipoprotein. An apparent molecular weight of 15,000 for this protein was determined from amino acid analysis and sodium dodecylsulfate/polyacrylamide gel electrophoresis. The amino acid composition, however, does not show similarities with the amino acid composition of the lipoprotein covalently linked to murein, which has a molecular weight of 7,300 as described previously in Proteus mirabilis.Abbreviation SDS sodium dodecylsulfate     

The role of chloroplast-membrane-protein synthesis in the circadian clock. Purification and partial characterization of a polypeptide which is suggested to be involved in the clock.

A polypeptide (polypeptide P39), which is presumed to involved in the photosynthetic circadian rhythm in the green alga Acetabularia, was purified from the EDTA-insoluble chloroplast membrane fraction by means of preparative dodecylsulfate gel electrophoresis and then partially characterized. The purity of the isolated polypeptide P39 was confirmed by a further electrophoresis on an analytical dodecylsulfate gel and further elucidated by amino-terminal analysis which shows that glycine is the only amino-terminal amino acid of the purified polypeptide material. The molecular weight of the polypeptide P39 was found to be about 39,000 on analytical gel electrophoresis and the value was further supported by those obtained from amino acid composition and peptide mapping. The amino acid composition of polypeptide P39 showed that the proportion of intermediate amino acid groups is high while the proportion of hydrophilic amino acid groups is well balanced by that of hydrophobic amino acid groups, a property characteristic of membrane proteins.     

Sugar transport by the bacterial phosphotransferase system. Fluorescence studies of subunit interactions of enzyme I

Enzyme I of the bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) exhibits a temperature-dependent monomer/dimer equilibrium. The accompanying paper (Han, M. K., Roseman, S., and Brand, L. (1990) J. Biol. Chem. 265, 1985-1995) shows that the C-terminal -SH residue (Cys-575) can be modified specifically with fluorescent probes such as pyrene maleimide. The derivative retains full enzyme activity, and is capable of forming dimers at room temperature. In the present studies, Enzyme I labeled in this way is found to exhibit a temperature-, concentration-, and pH-dependent monomer/dimer association. The kinetics of dimer formation of Enzyme I is measured in the following way. A derivatized Enzyme I sample is prepared with a pyrene moiety irreversibly attached to the C-terminal -SH residue and 5,5'-dithiobis-2-nitrobenzoic acid reversibly attached to the other 3 -SH residues. This modified enzyme does not form dimers at room temperature. Addition of dithiothreitol results in total release of the thionitrobenzoate anion within 2 min. After the three -SH groups are unblocked, steady-state and nanosecond time-resolved emission anisotropy measurements indicate the dimer is formed over a period of 30 min. In a similar experiment, little dimer formation is observed at 3 degrees C, at temperature at which the native enzyme also does not form dimers. Tryptophan fluorescence is also examined during the release of the thionitrobenzoate. After the completion of thionitrobenzoate release, additional slow steady-state tryptophan fluorescence changes are observed. These results suggest that dimer formation may be preceded by a conformational change following thionitrobenzoate release.     

A selenocysteine variant of the human copper chaperone for superoxide dismutase. A Se-XAS probe of cluster composition at the domain 3-domain 3 dimer interface

We report the semisynthesis of a selenocysteine (Sec) derivative of the human copper chaperone for superoxide dismutase, substituted with Sec at the C-terminal C246 residue. Measurements of hCCS-induced SOD1 activation were used to show that the C-terminal CXC sequence is both necessary and sufficient for EZn-SOD maturation. Therefore, an active CAU variant carrying Sec as the terminal amino acid was prepared by expressed protein ligation of a single selenocysteine amino acid to a 243-CA truncation. This reaction proceeded in high yield and generated the desired 243-CAX (X = C or U) protein with the expected mass. Se-edge XAS of the apoprotein indicated that both Se-S and Se-Se interactions were present in a 0.3:0.7 ratio, indicating an equilibrium between species with either a selenosulfide or a diselenide cross-link. After reduction on immobilized TCEP, the ligated Cys and Sec apoproteins bound up to 2.5 Cu(I) ions per hCCS monomer with both Cu and Se as constituent atoms of the cluster which forms at the domain 3 interface of a hCCS dimer. Merging of XAS data at the Cu and Se K-absorption edges provided additional details of the cluster composition, specifically the fact that both Se atoms occupied bridging positions between two Cu(I) atoms. Further, the requirement for identical Cu-Se bond lengths and Debye-Waller factors at each absorption edge allowed us to rule out simple models for the cluster composition such as a bis-Cys(Sec)-bridged dinuclear cluster and was indicative of a more complex cluster with a nuclearity of >or=3.     

Sulfhydryl-selective fluorescence labeling of lipoprotein(a) reveals evidence for one single disulfide linkage between apoproteins(a) and B-100.

Human lipoprotein(a) and low-density lipoprotein were labeled with two different sulfhydryl-selective fluorescence markers. The hydrophilic fluorophore lucifer yellow iodoacetamide and the apolar compound 6-acryloyl-2-(dimethylamino)naphthalene were used to derivatize free -SH groups in the lipoproteins. Three sulfhydryls could be detected in low-density lipoprotein, whereas only two cysteines were available in lipoprotein(a). One of the three -SH groups in low-density lipoprotein was shown to be located in close proximity to the particle surface. We suggest that this surface-exposed cysteine of apoprotein B-100 serves as a component for the disulfide linkage to apoprotein(a) in lipoprotein(a).     

Studies on plantacyanin. I. Distribution in the plant kingdom, subcellular localization and physicochemical properties

The standard preparative procedure used earlier for the isolation of plantacyanin from cucumber peelings and spinach leaves was employed for the detection of this protein in 15 plants. It was found that four of these 15 species of plants contain plantacyanin. From the study of the subcellular distribution of plantacyanin in spinach leaves, it has been concluded that the protein is associated with chloroplasts. Macromolecular properties as well as optical, fluorescence and EPR spectra of plantacyanin from cucumber were studied. It has been shown that at alkaline pH, a short living state of the protein active site is formed. This state (state 3) has an absorption band at 575 nm. The incubation of the protein in alkaline media under anaerobic conditions brings about the bleaching of plantacyanin accompanied by the disappearance of the EPR signal. The effect of some protein-modifying agents was studied and the data obtained are discussed from the point of view of possible ligand amino acids of copper in plantacyanin. The protein copper can be removed by diethyldithiocarbamate and the procedure of reconstitution of holoprotein from apoprotein and Cu2+ was developed.     

Mitochondrial adenylate kinase from chicken liver. Purification characterization and its cell-free synthesis

Mitochondrial adenylate kinase has been purified 5400-fold from chicken liver extract in an overall yield of 36%. The purified enzyme has a specific activity of 810 U/mg, a molecular weight of 28 000, and the following amino acid composition: 21 aspartic acid or asparagine, 14 threonine, 17 serine, 27 glutamic acid or glutamine, 16 proline, 22 glycine, 22 alanine, 15 valine, 6 methionine, 11 isoleucine, 29 leucine, 5 tyrosine, 7 phenylalanine, 16 lysine, 7 histidine, 19 arginine, 3 half-cystine, and no tryptophan, totalling 257 residues. The purified enzyme has one disulfide bond and one sulfhydryl group. The disulfide bond is related to the active conformation of the enzyme, whereas the sulfhydryl group does not contribute to the enzyme activity. The sulfhydryl group is easily oxidized in the presence of Cu2+ resulting in the formation of dimer with about one half of the specific activity of the monomer. The enzyme is similar to porcine heart mitochondrial adenylate kinase in antigenicity but different from chicken cytosolic adenylate kinase. Mitochondrial adenylate kinase was synthesized in the mRNA-dependent rabbit reticulocyte lysate system programmed with total chicken liver RNA. The mobility in sodium dodecylsulfate gel electrophoresis of the product obtained in vitro was the same as that of the purified mitochondrial adenylate kinase. This evidence indicates that the mitochondrial adenylate kinase is synthesized as a polypeptide with a molecular weight indistinguishable from that of the mature protein.