Topography of the C terminus of cytochrome b5 tightly bound to dimyristoylphosphatidylcholine vesicles

Cytochrome b5 holoenzyme was bound asymmetrically in the tightly bound form to small unilamellar dimyristoylphosphatidylcholine vesicles. [3H]Taurine, a membrane-impermeant nucleophile, was added to the external medium and was then cross-linked to cytochrome carboxyl residues by the addition of a water-soluble carbodiimide. Nonpolar peptide was isolated after trypsin digestion of taurine-labeled apocytochrome b5 and contained 1.7-1.9 residues of taurine. The C-terminal tetrapeptide containing residues Thr130-Asn133 was generated by chymotryptic hydrolysis of radiolabeled nonpolar peptide and was purified by gel filtration and ion exchange chromatography. Amino acid analysis of the C-terminal tetrapeptide showed that about 1.6 mol of taurine was cross-linked per mol of peptide. When the experiment was performed with taurine trapped inside the vesicles, no cross-linking was observed. The results suggest that when cytochrome b5 holoenzyme is bound to vesicles in the tight binding form, the C terminus is located on the external surface of the vesicles.     

Structure-activity relationship study of human interleukin-3: role of the C-terminal region for biological activity.

A structure-activity relationship study of human interleukin-3 (huIL-3) was performed by functional analysis of huIL-3 deletion and substitution variants combined with epitope mapping of huIL-3 specific neutralizing monoclonal antibodies (mAb). Analysis of the huIL-3 variants was accomplished by defining their capacity to compete with wild-type huIL-3 for binding to the huIL-3 receptor and to induce the proliferation of the huIL-3 dependent cell line M-O7. HuIL-3 variants with either 14 amino acids (aa) deleted from the N-terminus or eight aa from the C-terminus retained full biological activity in vitro. An huIL-3 variant, with 18 N-terminal aa deleted, exhibited a greater than 7-fold reduced receptor binding capacity and proliferative activity. No biological activity could be detected with a variant where 22 C-terminal aa have been deleted. Neutralizing mAb recognizing presumed discontinuous epitopes failed to interact with the latter deletion variant indicating a possible location of their epitopes within the C-terminal region. Computer-aided structure prediction and sequence homology analysis of this region indicated the presence of an amphiphilic alpha-helix with highly conserved residues like Lys110 and Leu111. Substitution of Lys110 with either Glu or Ala resulted in variants with a 10-fold reduced activity in the receptor binding assay and the proliferation assay. Further variants, where Leu111 was substituted by Pro or Met, were totally inactive in these assays. Analysis of the binding of the two neutralizing mAb to these substitution variants showed that they did not bind to either of the Leu111 variants suggesting that Leu111 is part of an active site. Based on our results, a possible model for the structure of the huIL-3 molecule can be constructed with two active sites in close proximity.     

NADH binding to cytochrome b5 reductase blocks the acetylation of lysine 110

Lysine residues outside of the NADH-binding site in the soluble catalytic fragment of cytochrome b5 reductase were modified with ethyl acetimidate and acetic anhydride while the binding site was protected by formation of the stable oxidized nucleotide-reduced flavoprotein complex. This treatment had a minimal effect on enzyme activity; the turnover number with potassium ferricyanide was 45,300 in the native reductase and 39,200 in the derivative. Subsequent reaction with [3H]acetic anhydride after the removal of NADH resulted in the loss of 91% of the enzyme activity and the incorporation of 1.9 eq of acetyl groups into the protein. Treatment with 1 M hydroxylamine at pH 13 indicated that only lysine residues were acetylated, and fragmentation of the derivative with cyanogen bromide and subfragmentation with trypsin and chymotrypsin demonstrated that only Lys110 was labeled at high specific activity, with a stoichiometry of 0.83 acetyl groups/mol, in good agreement with the loss of enzyme activity observed. The remaining label was distributed at low levels among four or more additional lysine residues. These results demonstrate that only Lys110 is specifically protected by NADH and is therefore the residue which provides the epsilon-amino group implicated in NADH binding in cytochrome b5 reductase.     

Construction and sequence of cDNA for rat liver stearyl coenzyme A desaturase

Hepatic poly(A+) RNA from rats induced for stearyl-CoA desaturase was used for primer-extension of cDNA coding for stearyl-CoA desaturase. Previously, Northern blot analysis showed that translatable desaturase mRNA is 4,900 nucleotides in length (Thiede, M. A., and Strittmatter, P. (1985) J. Biol. Chem. 260, 14459-14463). Six overlapping cDNAs, ranging from 850 to 1450 bases, were used to compile the 4,689-nucleotide sequence. The cDNA includes a 1,074-base open reading frame coding for 358 amino acids, corresponding to a molecular mass of 41,400 daltons. Positive identification of this open reading frame was accomplished by matching the amino acid sequence of both amino-terminal and cyanogen bromide peptides of the purified enzyme with regions of the sequence deduced from the cDNA. Amino acid composition data from the cDNA compares well with that from the desaturase. The protein contains 62% hydrophobic amino acids. An interesting feature of this mRNA is the 3,500-base 3' noncoding region, which has been localized on a single 3' exon by Southern blot analysis.     

Complete amino acid sequence of steer liver microsomal NADH-cytochrome b5 reductase

The complete covalent structure of liver microsomal NADH-cytochrome b5 reductase from steer liver microsomes was determined. Cleavage at methionyl bonds gave 10 peptides accounting for all the residues of the protein. Acid cleavage of the reductase at the Asp-Pro bonds gave three peptides accounting for all the CNBr peptides in the molecule. Subfragmentation of these peptides by chemical and enzymatic cleavage provided overlaps which established all the fragments in an unambiguous sequence of 300 residues, corresponding to Mr 34,110. Limited tryptic digestion cleaved reductase at residues 28 and 119, yielding a preparation having two noncovalently linked peptides having a conformation which binds flavin and retains the structural features essential for NADH-cytochrome b5 activity. A model for the secondary structure of cytochrome b5 reductase is proposed that is based on computer-assisted analysis of the amino acid sequence. In this model the beta-turns are predominant and there is some 25% alpha and 30% beta structure.     

3H]Captopril binding to membrane associated angiotensin converting enzyme

[3H]Captopril binding to membrane fractions of rat tissues is saturable and reversible with a KD of 2.4 nM. [3H]Captopril binding and angiotensin converting enzyme measured with hippuryl-L-histidine-L-leucine are distributed in parallel between different tissues and brain regions, with highest levels in the choroid plexus, lung and corpus striatum. Captopril, N-(1(S)-carboxy-3-phenyl-propyl)-L-alanyl-L-proline, N-(1(S)-carboxy-3-phenyl-propyl)-L-lysyl-L-proline, teprotide, thiorphan and S-acetylcaptopril each have similar potencies for inhibition of [3H]captopril binding and of angiotensin converting enzyme. These data strongly indicate that [3H]captopril binds selectively to angiotensin converting enzyme. [3H]Captopril binding evaluation should help clarify the localization and function of angiotensin converting enzyme and assist in defining pharmacologic actions of captopril.     

Nontoxic lipopolysaccharide from Rhodopseudomonas sphaeroides ATCC 17023.

Chemical analysis of the lipopolysaccharide from Rhodopseudomonas sphaeroides ATCC 17023, isolated by the phenol-chloroform-petroleum ether method, revealed the presence of glucuronic acid, 2-keto-3-deoxyoctonate, threonine, and phosphorus in the polysaccharide moiety. The lipid A component contained glucosamine, glucosamine phosphate, amide-bound 3-oxotetradecanoic acid and 3-hydroxytetradecanoic acid, and ester-bound 3-hydroxydecanoic acid and 7-tetradecenoic acid. Structural similarity of the lipid A from R. sphaeroides ATCC 17023 to enterobacterial lipid A is indicated by the existence of a serological cross-reaction occurring between the lipid A from R. sphaeroides ATCC 17023 and that from Salmonella minnesota R595. The lipopolysaccharide and lipid A of R. sphaeroides, however, were found to be neither toxic in mice nor pyrogenic in rabbits.     

Cotranscription and processing of 23S, 4.5S and 5S rRNA in chloroplasts from Zea mays

The termini of rRNA processing intermediates and of mature rRNA species encoded by the 3' terminal region of 23S rDNA, by 4.5S rDNA, by the 5' terminal region of 5S rDNA and by the 23S/4.5S/5S intergenic regions from Zea mays chloroplast DNA were determined by using total RNA isolated from maize chloroplasts and 32P-labelled rDNA restriction fragments of these regions for nuclease S1 and primer extension mapping. Several processing sites detectable by both 3' and 5' terminally labelled probes could be identified and correlated to the secondary structure for the 23S/4.5S intergenic region. The complete 4.5S/5S intergenic region can be reverse transcribed and a common processing site for maturation of 4.5S and 5S rRNA close to the 3' end of 4.5S rRNA was detected. It is therefore concluded that 23S, 4.5S and 5S rRNA are cotranscribed.