Assay of mouse liver uroporphyrinogen decarboxylase by reverse-phase high-performance liquid chromatography

A method for the estimation of hepatic uroporphyrinogen decarboxylase activity employing reverse-phase HPLC is described. Mouse liver homogenate in 0.25 M sucrose was pretreated with a suspension of cellulose phosphate and then centrifuged to remove hemoglobin and debris. The supernatant was used as the enzyme source. Incubations were acidified, oxidized, and centrifuged only before analysis of the porphyrins formed, using a Spherisorb ODS column and a gradient solvent system constructed from methanol/lithium citrate mixtures. Coproporphyrinogen formation by BALB/c mouse liver supernatant was estimated as about 5.0 and 9.1 pmol/min/mg protein from uroporphyrinogens I and III, respectively, at 10 microM substrate concentration and pH 6.8. Decarboxylation of pentacarboxyporphyrinogens (the last step in coproporphyrinogen formation) proved to be easily measured. Coproporphyrinogen formation from pentacarboxyporphyrinogen III abd (20 microM) at pH 6.8 was about 109 pmol/min/mg protein. Pentacarboxyporphyrinogen I was not as good a substrate as III abd but was decarboxylated faster at pH 5.4 than at 6.8, and at the lower pH and at 10 microM concentration of substrate 42 pmol of coproporphyrinogen was formed/min/mg protein. These results compared favorably with those obtained by previously published procedures involving time-consuming extraction and esterification steps.     

The covalent structure of cartilage collagen. Amino acid sequence of residues 552–661 of bovine α1(II) chains

The covalent structure of the first 111 residues from the N-terminus of peptide α1(II)-CB10 from bovine nasal-cartilage collagen is presented. This region comprises residues 552–661 of the α1(II) chain. The sequence was determined by automated Edman degradation of peptide α1(II)-CB10 and of peptides produced by cleavage with trypsin and hydroxylamine. Comparison of this region of the α1(II) chain with the homologous segment of the α1(I) chain indicated a homology level of 85%, slightly higher than that of 81% reported for the N-terminal region of the α1(II) chain (Butler, Miller & Finch (1976) Biochemistry 15, 3000–3006). The occurrence of two residues of glycosylated hydroxylysine was established at positions 564 and 603, the first present exclusively as galactosylhydroxylysine and the latter as a mixture of galactosylhydroxylysine and glucosylgalactosylhydroxylysine. Also, two residues at positions 648 and 657 were tentatively identified as glycosylated hydroxylysines. The amino acid sequences adjacent to the hydroxylysine residues so far identified in the α1(II) chain were compared with the homologous regions of the α1(I) and α2 chains, but no obvious prerequisite for hydroxylation could be seen. From comparison with the homologous sequence of the α1(I) chain, it appears that the α1(II)-chain sequence presented here contains three more amino acids than that reported for the α1(I) chain. This triplet would be interposed between residues 63 and 64 of the reported sequence of peptide α1(I)-CB7 from calf skin collagen. Data on the purification of the subpeptides and their amino acid compositions have been deposited as Supplementary Publication SUP 50087 (7 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

EPR determination of the oxidation-reduction potentials of the hemes in cytochrome c3 from Desulfovibrio vulgaris.

EPR spectroscopy in conjunction with oxidation-reduction potentiometry has been used to determine the half-reduction potentials of the four hemes of cytochrome c3. As predicted, the four hemes of cytochrome c3 have different mid-point potentials. The Em values are: Heme I,--284 mV; Heme II,--310 mV; Heme III,--324 mV and Heme IV,--319 mV. The n-values in each case was near one.     

Distribution and partial purification of a liver membrane protein capable of inactivating cytosol enzymes.

1. The inactivation of cytosol enzymes in liver extracts was carried out by several subcellular fractions, with plasma membranes having the highest specific activity. Rough and smooth microsomal fractions were both active, whereas lysosmal inactivation capacity appeared to be derived entirely from contaminating plasma-membrane fragments. 2. Inactivation capacity in liver fractions was derived from parenchymal cells. Of the non-liver cells tested, plasma membranes from H35 hepatoma cells were able to inactivate glucose 6-phosphate dehydrogenase (EC 1.1.1.49), adipocyte "ghosts" showed slight activity and erythrocyte and reticulocyte "ghosts" were inactive. 3. Liposomes prepared from pure lipids with net negative, positive or neutral charge did not possess inactivation capacity. 4. Liver plasma-membrane inactivation capacity was destroyed by heating at 50 degrees C. 5. Inactivation factor solubilized from membranes by trypsin plus Triton X-100 treatment was partially purified by (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography and hydroxyapatite chromatography. 6. Partially purified inactivation factor analysed by gel electrophoresis gave a major protein band that co-migrated with capacity for inactivation of glucose 6-phosphate dehydrogenase. 7. It is concluded that inactivation factor is a membrane protein whose intracellular distribution and other properties are consistent with a possible role for this activity in the initial step of protein degradation.     

The use of specific lysine modifications to locate the reaction site of cytochrome c with sulfite oxidase

The reduction of cytochrome c by beef liver sulfite oxidase was found to be strongly inhibited by high ionic strength, indicating the importance of electrostatic interactions to the reaction. The reaction rates of sulfite oxidase with singly trifluoroacetylated or trifluoromethylphenylcarbamylated cytochrome c derivatives were studied to determine the role of individual lysines in the reaction. The reaction rate was decreased by modification of the lysines immediately surrounding the heme crevice, the decreases following the order: Lys 13 > Lys 25 Lys 79 ≈ Lys 87 > Lys 8 ≈ Lys 27 ≈ Lys 72. Modification of lysines 22, 55, 88, 99, and 100 had no effect on the reaction rate. These results indicate that the interaction site on cytochrome c for sulfite oxidase is at the heme crevice region, and overlaps considerable with that for cytochrome oxidase.     

Detection of low molecular weight copper(II) and zinc(II) binding ligands in ultrafiltered milks—the citrate connection

Low molecular weight zinc(II) and copper(II) binding ligands were detected in ultrafiltered human, bovine, and goat milk by the application of the method of modified gel chromatography. Human milk contains at least three detectable low molecular weight copper binders, whereas bovine and goat milk contain at least two. All three milks show two copper binding peaks with the same elution volumes. Zinc chromatograms were less specific than copper. Zinc showed only a single detectable low molecular weight binding ligand common to all three milks. Elution volumes for both zinc(II) and copper(II) citrate and picolinate systems were measured. Elution volumes of both copper(II) and zinc(II) citrate complexes are identical to elution volumes of an intense peak observed with all three milks; it is reasonable to assume that at least part of this peak corresponds to citrate. Human milk alone has a relatively intense binding peak for copper(II) at the same elution volume as the glutamate complex. Human and goat milk have another low intensity copper(II) binding ligand peak at the same elution volume; a number of amino acid complexes have binding peaks at this position. No peak characteristic of the zinc(II) or copper(II) picolinate systems could be found with any of the milks.