On the oligosaccharide moiety of angiotensin-converting enzyme.

Two glycopeptide fractions in a pronase digest of rabbit pulmonary angiotensin-converting enzyme were resolved by gel filtration. GP-I, the minor component (～1 mole/mol enzyme) contained mannose, galactose, glucose $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and sialic acid in an approximate molar ratio of 1:5:3:4:1:2 and molar equivalents of aspartic acid, threonine and serine. GP-II, the major oligosaccharide unit (～ 12 moles/mol enzyme, ～ 90% of total carbohydrate), contained fucose, mannose, galactose, $\text{N}$-acetylglucosamine, sialic acid and aspartic acid in a molar ratio of 1:4:4:4:1:1. Although accounting for about one-quarter of the weight of the enzyme, GP-II did not compete with the intact glycoprotein for binding to goat antienzyme antibodies. Some structural features of GP-II were deduced by periodate oxidation and digestion with various glycosidases.     

Geometrical and conformational preferences of the 9-fluorenylmethoxycarbonyl-amino moiety.

Structural parameters, originating from x-ray crystallographic data, have been compiled for 13 derivatives of amino acids, peptides and related compounds, which contain a total of 14 Fmoc-NH- moieties. For these moieties, molecular geometries and conformations--described by the omegao, theta1, theta2 and theta3' torsion angles--were analysed and compared with the corresponding parameters for the Z-NH- and Boc-NH-moieties (290 and 553, respectively). To gain a deeper insight into the conformational features of the Fmoc-NH- moiety, ab initio free molecule calculations were performed for fully relaxed minima. Also the potential energy surface as a function of the torsion angles (theta3', theta2) was generated. The conformational features of the Fmoc-NH- moiety: (i) two possible values for the angle omegao (approximately 180 degrees or, rarely, approximately theta degrees) and (ii) the angle theta1 = 180 degrees +/- 15 degrees, are common to the Z-NH- and Boc-NH- systems. By contrast, the theta2 and theta3 angles in the Fmoc, Z and Boc groups differ essentially. In the Fmoc groups theta2 mostly has values of 180 degrees +/- 30 degrees and values up [115 degrees] seem to be forbidden, whereas fewer than half of the Z groups adopt theta2 approximately 180 degrees and the remainder have theta2 in the range of [90 degrees +/- 20 degrees]. On the other hand, the Boc methyl groups are staggered. The theta3 values observed for Fmoc are limited to the regions of 180 degrees +/- 20 degrees and 160 degrees +/- 20 degrees], while for the Z group a variety of theta3 occurs. The orientation of the fluorenyl vs the urethane function is mostly trans. Our results suggest a lower conformational flexibility for the Fmoc group compared with that of the Z group. Our calculations confirm that the observed conformational features for the Fmoc-NH- moiety are inherent properties. The Fmoc-NH-moiety in crystals involves the participation of its O=C-NH functionality in hydrogen bonds.     

Specific maltose labeling in the reducing glucose moiety.

Radioactive maltose with label in the reducing glucose moiety was prepared using a glucosyltransferase enzyme to catalyze exchange of [6-³H]glucose into unlabeled maltose. The enzyme was isolated from spinach by ammonium sulfate precipitation followed by DEAE column chromatography. A 77% yield of [6-³H]maltose was obtained after a reaction of 100 nmol of maltose with 0.0147 nmol of [6-³H]glucose was catalyzed by the most active column peak. The product was exclusively labeled in the reducing glucose moiety as indicated by the label occurring only in sorbitol following sodium borohydride reduction and sulfuric acid hydrolysis. Between 88.3 and 96.0% of the tritium in the synthesized preparation was present as [6-³H]maltose by Dowex 1-X4 chromatography. This column separates [6-³H]maltose-[U-¹⁴C]maltose mixtures and [6-³H]glucose-[U-¹⁴C]glucose mixtures apparently as a result of an isotope effect.     

Production of a precursor to the pyrimidine moiety of thiamine.

The supernatant fluid from cultures of Escherichia coli W-11, a pur E mutant, prevented the inhibition of growth of E. coli B in a medium containing adenine or adenosine. Adenine inhibition was prevented more readily than adenosine inhibition. More than 90% of the biological activity of the supernatant fluid was recovered in the anionic fraction after treatment with Dowex-50 (NH4+). The cationic fraction, containing large amounts of 5-aminoimidazole ribonucleoside (AIRS), did not prevent adenine inhibition. The W-11 supernatant fluid was shown by bioautography to contain only one compound that prevented adenine inhibition. Proliferating and non-proliferating cultures produced only one compound that prevented adenine inhibition. The compound was shown to be an intermediate (int-1) in the biosynthesis of the pyrimidine moiety of thiamine, Int-1 was stable during sterilization at 121 C for 15 min, during concentration by either flask evaporation or lyophilization, and after storage for several days at 4 C or at -- 20 C. Int-1 was distinguishable from other known derivatives or intermediates of the pyrimidine moiety. A scheme is presented that illustrates the proposed relationship between int-1 and the synthesis of thiamine.     

The mechanism of peroxidase-mediated cytotoxicity. II. Role of the heme moiety

Various peroxidases in the presence of hydrogen peroxide and a halide ion have been shown to exert a cytolytic activity against erythrocytes and other cells. However, few studies have been done to elucidate the active site on the enzymes that is responsible for the cytotoxic activity. In addressing this question we found that boiling of horseradish peroxidase only partially abolishes its cytotoxic activity, suggesting that an intact tertiary structure of the protein may not be essential for the cytotoxic activity. This conclusion was confirmed by demonstrating that microperoxidase, hemin, and hematoheme also exert cytotoxic activity in the presence of hydrogen peroxide and iodide, the kinetics of which were identical to those obtained with the peroxidases. Fluoride, bromide, and thiocyanate could not replace iodide in any of these systems. These results indicate that the active site for the cytotoxic activity of the peroxidases is located within the heme moiety, whereas the protein portions of the enzymes affect the cytotoxic activity of the enzymes only in an indirect manner. We also tested a variety of compounds for their ability to inhibit the cytolytic reaction toward erythrocytes. We found that compounds such as thiourea, thionicotinamide, and uric acid are much more potent inhibitors of the cytolytic reaction than tyrosine and histidine. These observations support the concept that oxidative reactions rather than halogenation reactions are the primary cause of the peroxidase-mediated lysis of erythrocytes.     

31P-NMR study of the phospholipid moiety of lipophorin subspecies.

31P-NMR spectra of four distinct subspecies of Manduca sexta hemolymph lipophorin revealed the presence of two resonances separated by 0.6 ppm. Phospholipid analysis of the lipoproteins showed that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were present and their mass ratio correlated well to the intensity of the two resonances in each of the different subspecies. The two resonances persisted in 31P-NMR spectra of organic solvent extracts of lipophorin. These results, together with the fact that PE, but not PC, can form an intramolecular hydrogen bond between the phosphate oxygen and the amino group of ethanolamine, resulting in deshielding of the phosphorus nucleus (and a 0.6 ppm downfield shift), strongly suggest the resonances observed represent the PC and PE components of these lipoproteins. 31P-NMR line-width data obtained as a function of temperature and solvent viscosity were used to calculate the chemical shift anisotropy (delta sigma), intrinsic viscosity (eta'), and lateral diffusion coefficients (DT) of PC and PE in different lipophorin subspecies. eta' and DT for PC and PE were similar among high-density lipophorins but differed in low-density lipophorin (LDLp). These differences may be related to the large increase in diacylglycerol content in this particle and/or the association of up to 16 molecules of apolipophorin III. On the basis of the known lipid compositional differences between LDLp and high-density lipophorin subspecies, we propose that uptake of large amounts of diacylglycerol during LDLp formation results in partitioning of this lipid to the surface monolayer where it intercalates between phospholipid molecules. Diacylglycerol intercalation creates gaps between phospholipid head groups that expose the hydrophobic surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of chloramphenicol. Studies on the origin of the dichloroacetyl moiety

Chloramphenicol produced by cultures of Streptomyces species 3022a supplemented with sodium [1,2-13C]acetate was labelled with 13C exclusively in the dichloromethine (2.6 +/- 0.1%) and carbonyl (0.59 +/- 0.05% carbon atoms. Satellite signals from 13C-13C coupling between covalently bonded 13C-enriched carbon atoms were too intense to be attributed to random combination of labelled atoms at the average enrichments measured, but their intensity relative to those of the signals for uncoupled 13C atoms indicated that most of the precursor had been incorporated after 13C-13C bond fission. Since [2,3-13c]succinic acid enriched only the carbonyl carbon atom of chloramphenicol, these results suggest that neither acetate nor a Krebs cycle intermediate is a direct precursor of the dichloroacetyl group. Cultures supplemented with [2-3h]-or [2h2]-dichloroacetic acid incorporated negligible amounts of isotope into the antibiotic; on this evidence, the free acid is not an intermediate in chloramphenicol biosynthesis and the acylation step may precede chlorination.     

Degradation of tetrahydrofurfuryl alcohol by Ralstonia eutropha is initiated by an inducible pyrroloquinoline quinone-dependent alcohol dehydrogenase.

An organism tentatively identified as Ralstonia eutropha was isolated from enrichment cultures containing tetrahydrofurfuryl alcohol (THFA) as the sole source of carbon and energy. The strain was able to tolerate up to 200 mM THFA in mineral salt medium. The degradation was initiated by an inducible ferricyanide-dependent alcohol dehydrogenase (ADH) which was detected in the soluble fraction of cell extracts. The enzyme catalyzed the oxidation of THFA to the corresponding tetrahydrofuran-2-carboxylic acid. Studies with n-pentanol as the substrate revealed that the corresponding aldehyde was released as a free intermediate. The enzyme was purified 211-fold to apparent homogeneity and could be identified as a quinohemoprotein containing one pyrroloquinoline quinone and one covalently bound heme c per monomer. It was a monomer of 73 kDa and had an isoelectric point of 9.1. A broad substrate spectrum was obtained for the enzyme, which converted different primary alcohols, starting from C2 compounds, secondary alcohols, diols, polyethylene glycol 6000, and aldehydes, including formaldehyde. A sequence identity of 65% with a quinohemoprotein ADH from Comamonas testosteroni was found by comparing 36 N-terminal amino acids. The ferricyanide-dependent ADH activity was induced during growth on different alcohols except ethanol. In addition to this activity, an NAD-dependent ADH was present depending on the alcohol used as the carbon source.     

Orientation of the rhodopsin sugar moiety in bovine disk membrane.

Rhodopsin from the bovine rod outer segment contains a covalently linked carbohydrate moiety (Heller, J. & Lawrence, M.A. (1973) Biochemistry 9, 864--868). We studied the location of this carbohydrate moiety on the disk membrane by using ferritin-conjugated concanavalin A and concanavalin A labelled with fluorescein isothiocyanate. Electron microscopic observation of sonicated disk membrane that was labelled with ferritin-concanavalin A revealed the electron-dense image of ferritin on the inner surface of the disk membrane and not on its outer surface. Intact disk membrane that was similarly treated with ferritin-concanavalin A showed a complete absence of ferritin molecules on its surface. In an independent series of experiments we confirmed that the sonicated disk membrane bound three to five times more fluorescein-labelled concanavalin A than the intact disk membrane did. From these experiments we conclude that the carbohydrate moiety of bovine rhodopsin is located on the inner surface of the disk membrane, in agreement with the report by Rohlich on the frog rod outer segment disk membrane (Rohlich, P. (1976) Nature 263, 789--791).     

Role of carbohydrate moiety in carboxypeptidase Y: structural study of mutant enzyme lacking carbohydrate moiety.

To study the roles of the carbohydrate moiety in the function of carboxypeptidase Y, asparagine residues at 13, 87, 168, and 368, the four-consensus N-linked glycosylation sites, were altered to alanine with site-directed mutagenesis. The mutant enzyme of 51 kDa completely lost the carbohydrate moiety which was present in the 61-kDa wild-type enzyme. Structural studies of the mutant enzyme showed that it maintained the native-like structure; hydrolytic activity, and substrate specificity of the mutant enzyme analogous to those of the wild-type enzyme. Susceptibility of the mutant enzyme toward proteolysis and pressure denaturation was reduced by 10-20%. It is concluded that the carbohydrate moiety functions to maintain the structural integrity of the enzyme under stressed.     

Nickel cytochrome c.Effect of protein moiety on the metal ion coordination.

Nickel cytochrome c has been synthesized by the reaction of metal-free porphyrin cytochrome c with Ni(II) ions in 0.6 Mglycylglycine and 4 M KSCN. Electronic spectra and susceptibility measurement showed the nickel to be in a high-spin octahedral configuration exemplifying the strong influence of the protein moiety as a macrocyclic ligand on the coordination chemistry of the metal ion. Nickel cytochrome c has the same electrophoretic mobility, helicity and pK values of conformational transitions as the native enzyme. At high pH, the partially denatured nickel cytochrome c becomes dimeric. Nitric oxide reacts with nickel cytochrome c to form the nitrosyl derivative with (formula: see text). Reaction of NO with nickel protoporphyrin IX dimethyl ester in toluene, pyridine, or methylthioethanol produced no stable nitrosyl products, clearly demonstrating the effect of protein on metal ion ligation.     

Carbohydrate moiety of immunoglobulins in health and pathology.

Most of glycoproteins described so far, including immunoglobulins, are glycosylated during post-translational modifications of protein molecules. Current knowledge of the structure of sugar chains in immunoglobulin molecules and their biological role in health and pathology is reviewed.