The isolation of bovine-heart cytochrome c oxidase subunits. Dependence on phospholipid and cholate-content.

The polypeptide chains of bovine-heart cytochrome c oxidase were preparatively isolated by a simple large-scale procedure based on gel permeation chromatography in the presence of sodium dodecyl sulphate. The resolution of the subunits as a function of the cholate and phospholipid content of the preparation was investigated. Cholate, and to a lesser extent, phospholipids interfere with the separation of the subunits; however, they do not prevent dissociation of the enzyme by SDS. Bovine-heart cytochrome c oxidase consists of six major subunits (estimated molecular weights in thousands: 40, 25, 20, 14, 12 and 10). In addition, the enzyme preparation contains at least five minor constituents, present in less than stoichiometric amounts. The first two of the three large subunits, all of which are hydrophobic, have amino-terminal N-formylmethionine. Subunit III, however, has a free methionine N-terminus.

Determination of the sequence which spans the beginning of the insertion region in Anacystis nidulans flavodoxin.

Anacystis nidulans flavodoxin is one of the long chain flavodoxins (Mayhew & Ludwig, 1975). Comparisons of its structure with the structures of shorter chain species (main text: Ludwig et al., 1982) show that in A. nidulans flavodoxin most of the extra residues occupy a region adjoining the third helix and the fifth strand of parallel sheet. The sequences of peptides isolated after cyanogen bromide cleavage and after digestion with Staphylococcus aureus protease, reported here, fit into the electron density map of A. nidulans flavodoxin, starting near the middle of the third helix, and verify that the major insertion interrupts the fifth strand of parallel sheet.

A comparative study of bark lectins from three elderberry (Sambucus) species.

Three elderberry lectins isolated from the bark of three different species of the genus Sambucus which are native to Europe (S. nigra), North America (S. canadensis), and Japan (S. sieboldiana) were studied comparatively with regard to their carbohydrate binding properties and some structural features. All three lectins contained two identical carbohydrate binding sites per molecule and showed a very high specificity for the Neu5Ac(alpha 2-6)-Gal/GalNAc sequence. However, relative affinities for various oligosaccharides were significantly different among them, suggesting differences in the detailed structure of the carbohydrate binding sites of these lectins. The three lectins were immunologically related, but not identical, and all were composed of hydrophobic and hydrophilic subunit regions, although the molecular sizes of these subunits were slightly different among the three lectins. N-terminal sequence analysis of the subunits of these lectins suggested that they have a very similar structure in this region but also indicated the occurrence of N-terminal processing such as the deletion of several amino acid residues at the N-termini for both hydrophobic and hydrophilic subunits of all three lectins. Tryptic peptide mapping of the three lectins showed a similar pattern for all of them but also showed the presence of some unique peptides for each lectin.

Reverse-phase high-performance liquid chromatography of hydrophobic proteins and fragments thereof.

Reverse-phase high-performance liquid chromatography (HPLC) resolution and recovery of cytochrome P-450 and bovine rhodopsin, both integral membrane proteins, and large peptides derived from P-450 LM2 were enhanced by utilizing ternary solvents. Surprisingly, most test materials eluted later in the gradient when using mixtures of acetonitrile and propanol in the mobile phase compared to using either solvent alone. Of the supports tested, the best recovery of hydrophobic cytochrome P-450 LM4 was experienced on the less retentive CN-bonded phase. Two alternate solvents for HPLC of polypeptides are proposed: (1) 0.02-0.1 M hexafluoroacetone/NH3, pH 7.2 for highly acidic peptides; and (2) 6 M formic acid/0.13 M trimethylamine, pH 1.5, vs 4 M formic acid/0.09 M trimethylamine in propanol for relatively insoluble peptides. Anomalous side reactions between formic acid and peptides can cause HPLC peak broadening, increased retention, and decreased resolution. These deleterious effects are thought to be due in part to formyl esterification of serine and threonine residues and appear to be reversible by aminoethanol treatment.

Amino acid sequence of cytochrome c from Tetrahymena pyriformis Phenoset A.

The cytochrome c of Tetrahymena pyriformis GL (Phenoset A) had an isoelectric point of 6.5 and by sequence the following composition: Asp(7) Asn(2) Thr(4) Ser(8) Glu(6) Gln(2) Pro(7) Gly(13) Ala(13) Val(7) Met(2) Ile(5) Leu(6) Tyr(2) Phe(5) Lys(11) His(3) Trp(1) Arg(3) Cys(2) (total 109 residues). The peptides derived from the protein afforded complete overlap, so a complete sequence could be determined without reference of homologous proteins. Alignment with other mitochondrial cytochromes c required two internal deletions totalling three residues and an N-terminal region two residues longer than, and a C-terminal region one residue shorter than, the previously known limits. The sequence was the most divergent of the known mitochondrial cytochromes c, suggesting a distant relationship of ciliates to other eukaryotes. Details of the sequence data have been deposited as Supplementary Publication no. SUP 50068 (37 pages) at The British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7 BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1976) 153,5.

Structural features of isozyme 2 of liver microsomal cytochrome P-450. Identification of a highly conserved cysteine-containing peptide.

Earlier findings in several laboratories have suggested that the various forms of cytochrome P-450 within a given species or from different species are not closely related. However, our present studies on the primary structure of the phenobarbital-inducible cytochrome from rabbit liver microsomes (P-450LM2) show that this protein is about 80% identical to the corresponding rat protein (P-450b), the sequence of which was recently deduced by others. For example, of the first 70 residues of the NH2-terminal sequences, 52 are identical, with most differences structurally conservative and attributable to point mutations. No evidence was found for heterogeneity in the region of the rabbit cytochrome corresponding to the variable region proposed for the rat cytochrome. A comparison of the known primary structure of P-450LM2 with that of P-450b and that of the bacterial camphor-hydroxylating cytochrome (P-450cam) as recently reported by others reveals only one highly conserved cysteine-containing peptide present in approximately the same position in each cytochrome. This peptide in the rabbit enzyme, -Arg-Ile-Gln-Glu-Glu-Ala-Arg-Cys147-Leu-Val-Glu-Glu-Leu-Arg-, and the corresponding peptides containing Cys152 in P-450b and Cys134 in P-450cam may serve an essential function such as providing the axial thiolate ligand to the heme iron atom.

Human hypoxanthine (guanine) phosphoribosyltransferase: an amino acid substitution in a mutant form of the enzyme isolated from a patient with gout.

We have investigated the molecular basis for a deficiency of the enzyme hypoxanthine (guanine) phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) in a patient with a severe form of gout. We reported in previous studies the isolation of a unique structural variant of HPRT from this patient's erythrocytes and cultured lymphoblasts. This enzyme variant, which is called HPRTLondon, is characterized by a decreased concentration of HPRT protein in erythrocytes and lymphoblasts, a normal Vmax, a 5-fold increased Km for hypoxanthine, a normal isoelectric point, and an apparently smaller subunit molecular weight. Comparative peptide mapping experiments revealed a single abnormal tryptic peptide in HPRTLondon. Edman degradation of the aberrant peptide from HPRTLondon identified a serine-to-leucine amino acid substitution at position 109. This substitution can be explained by a single nucleotide change in the codon for serine-109 (UCA leads to UUA). Thus a mutation at the HPRT locus has now been defined at the molecular level.

The activator protein for glucosylceramide beta-glucosidase from guinea pig liver. Improved isolation method and complete amino acid sequence.

beta-Glucosidase activator (SAP-2) is a family of heat-stable, acidic glycoproteins which stimulate enzymatic hydrolysis of glucosylceramide. In this study, we improved the purification method and found that SAP-2 is highly heterogeneous. A hot water extract of frozen guinea pig liver was fractionated by ammonium sulfate sedimentation, then chromatographed with DEAE-Sephacel, Sephadex G-75, and concanavalin A-Sepharose. A fraction binding to concanavalin A-Sepharose was purified further with a C4 high performance liquid chromatography reverse phase column. This yielded several peaks, the main one of which was studied. The specific activity of the purified SAP-2 was 35 units/micrograms (1 unit produces 50% stimulation of a basal glucosidase preparation). N-terminal amino acid sequencing showed that this preparation is a mixture of polypeptides differing in the presence or absence of one or two of the end amino acids. The complete amino acid sequence of the 81 residues in SAP-2 has been determined. Comparison of the sequence of guinea pig SAP-2 with the sequence of human sphingomyelinase activator revealed 58% homology and quite similar hydropathy profiles. Both proteins possess a highly hydrophilic region around Asn-22, which is glycosylated, and 6 cysteine residues, in oxidized form, located in the same positions. Comparison with the published nucleotide sequence for the precursor form of the human activator protein for sulfatide sulfatase (SAP-1) suggested that this activator also has a possibly glycosylated Asn and 6 Cys residues at similar positions, although the remainder of the molecule is somewhat different. Examination of another region of the precursor's nucleotide sequence, assuming a few changes in the identifications, revealed the presence of the sphingomyelinase activator. It appears that two or more activators are derived from a single precursor protein. Marked homologies were seen also with a lung surfactant protein and a sulfated glycoprotein from Sertoli cells.

Purification, physicochemical, and kinetic properties of liver acetyl-CoA:arylamine N-acetyltransferase from rapid acetylator rabbits.

Cytosolic liver acetyl-CoA:arylamine N-acetyltransferase (EC 2.3.1.5) from homozygous rapid acetylator rabbits (strain III/J) was purified to homogeneity as judged by gel filtration sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis and isoelectrofocusing. The isoelectric point was estimated to be 5.2. The molecular weight was determined to be 33,500 by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis and 33,000 by Sephacryl S-200 gel filtration. The amino acid composition is reported and 16 tryptic peptides were sequenced by Edmann degradation, including a peptide from which a very specific oligonucleotide probe can be synthesized. The enzyme contained neither amino sugars nor cofactors. A broad pH optimum from pH 5.9 to 8.6 was observed. N-Acetyltransferase activity showed a strong dependency on the salt concentration. From the influence of the basicity of the acceptor amine on the maximum velocity, it was concluded that the formation of the covalent acetyl-enzyme intermediate is the rate-limiting step in the N-acetyltransferase-catalyzed acetylation of amines. The covalent intermediate reacts, then, in a fast step with the acceptor amine, when using aniline derivatives with pKa values ranging from 5.65 to 1.74. However, with the weakly basic 4-nitroaniline, the acetyltransfer from the catalytic intermediate to the amine seems to be rate-limiting. A structure-activity study of 30 aniline derivatives that differ in hydrophobicity, position, size, charge, and number of substituents showed that some ortho-substituted derivatives were not acetylated.

Heterogeneity of amino acid sequence in hippopotamus cytochrome c.

The amino acid sequences of chymotryptic and tryptic peptides of Hippopotamus amphibius cytochrome c were determined by a recent modification of the manual Edman sequential degradation procedure. They were ordered by comparison with the structure of the hog protein. The hippopotamus protein differs in three positions: serine, alanine, and glutamine replace alanine, glutamic acid, and lysine in positions 43, 92, and 100, respectively. Since the artiodactyl suborders diverged in the mid-Eocene some 50 million years ago, the fact that representatives of some of them show no differences in their cytochromes c (cow, sheep, and hog), while another exhibits as many as three such differences, verifies that even in relatively closely related lines of descent the rate at which cytochrome c changes in the course of evolution is not constant. Furthermore, 10.6% of the hippopotamus cytochrome c preparation was shown to contain isoleucine instead of valine at position 3, indicating that one of the four animals from which the protein was obtained was heterozygous in the cytochrome c gene. Such heterogeneity is a necessary condition of evolutionary variation and has not been previously observed in the cytochrome c of a wild mammalian population.

Definition of cytochrome c binding domains by chemical modification. II. Identification and properties of singly substituted carboxydinitrophenyl cytochromes c at lysines 8, 13, 22, 27, 39, 60, 72, 87, and 99.

Sensitive thin layer peptide mapping is employed to establish the identity and the homogeneity of eight singly substituted 4-carboxy-2,6-dinitrophenyl derivatives of horse cytochrome c. Seven of the components, all of greater than 95% homogeneity, are modified at lysyl residues 13, 72, 87, 8, 27, 39, and 60. The eighth component is a mixture of derivatives at lysines 22 and 99. The positions of the modified residues were confirmed by the amino acid analysis and Edman sequential degradation of the CDNP-peptides. Physiochemical properties characteristic of cytochrome c are unchanged in the chemically modified products examined. These properties, that include the proton NMR spectrum, are sensitive probes of the polypeptide organization surrounding the heme prosthetic group. The lack of any discernable changes indicates that modification of the epsilon-amino groups on the surface of cytochrome c does not perturb the overall structure of the protein. The widespread distribution of the modifications on the surface of the molecule, together with the homogeneity and native conformation of the CDNP-derivatives make them well suited for assessing the effects of changes in the charge topography on the electron transfer activity of cytochrome c.

Purification and characterization of a unique isozyme of cytochrome P-450 from liver microsomes of ethanol-treated rabbits.

A new isozyme of cytochrome P-450 has been purified to electrophoretic homogeneity from hepatic microsomes of rabbits treated chronically with ethanol. Several criteria indicate that the ethanol-inducible cytochrome, which has a minimal molecular weight of 51,000 and is designated form 3a on the basis of its relative electrophoretic mobility, is distinct from the known isozymes of P-450. As judged spectrally, the new isozyme is high spin in the oxidized state, as is form 4, but differs in that the spin state is unperturbed by nonionic detergents. The absolute spectrum of the ferrous carbonyl complex of form 3a is red shifted as compared to that of forms 2, 3b, 3c, 4, and 6 and exhibits a maximum at 452 nm. The amino acid composition of form 3a is different from that of the other isozymes, and both the NH2- and COOH-terminal sequences are distinct; form 3a has an NH2-terminal alanine and a carboxyl-terminal leucine residue. Peptide mapping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following treatment with papain, chymotrypsin, or Staphylococcus aureus V8 protease and by high performance liquid chromatography following trypsinolysis indicates that form 3a is a unique gene product. This cytochrome displays the highest activity of all of the rabbit isozymes in the oxidation of ethanol to acetaldehyde and the p-hydroxylation of aniline when reconstituted with NADPH-cytochrome P-450 reductase and phospholipid in the presence of NADPH and oxygen.

Human hypoxanthine-guanine phosphoribosyltransferase. Complete amino acid sequence of the erythrocyte enzyme.

The entire amino acid sequence of hypoxanthine-guanine phosphoribosyltransferase from human erythrocytes has been defined. Peptide fragments formed by cleavage at arginine, glutamic acid, and methionine residues were analyzed by Edman degradation or digestion with carboxypeptidase. The complete primary structure of human hypoxanthine-guanine phosphoribosyltransferase was established by sequence analysis of 17 peptide fragments, 15 of which were purified by reverse-phase high pressure liquid chromatography. The enzyme is 217 residues long with a molecular weight equal to 24,470. Mass spectroscopy indicated that the NH2-terminal alanine is acetylated.

C-terminal ladder sequencing via matrix-assisted laser desorption mass spectrometry coupled with carboxypeptidase Y time-dependent and concentration-dependent digestions.

The utility of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for the analysis of C-terminal peptide ladders from carboxypeptidase Y (CPY) digestions is discussed. MALDI analysis of aliquots of an optimized time-dependent CPY digestion of ACTH 7-38 fragment allowed for the sequence of the first 19 amino acids from the C-terminus to be determined in 25 min of digestion time. A strategy for performing parallel concentration-dependent digestions on the MAL-DI plate is proven to be superior to the time-dependent approach as the method development time and practical amounts of both peptide and enzyme consumed are reduced significantly. The on-plate approach offered the same sequence information from the ACTH 7-38 fragment and was used to digest 22 peptides of various amino acid composition, size, charge, and polarity. Of the 22 peptides digested on-plate, sequence information was derived from 19 of them. A statistical analysis strategy for ladder sequencing utilizing t-statistics is offered as a method for placing confidence intervals on residue assignments.