Electron microscopic study of endogenous peroxidase activity in human liver macrophages

We evaluated the conditions of fixation for ultrastructurally demonstrating the endogenous peroxidase (PO) activity of macrophages in biopsied human liver. The application of microwaving and immersion fixation with tannic acid and aldehydes allowed excellent visualization of PO activity in the nuclear envelope (NE), rough endoplasmic reticulum (rER), and cytoplasmic granules (CG), with good preservation of cellular ultrastructures. The macrophages with PO activity showed one of the following five patterns of PO localization: positive in both the NE and rER but negative in the CG (type 1); negative in both the NE and rER but positive in the CG (type 2); negative in the NE but positive in both the rER and CG (type 3); positive in all three (type 4); PO negative (type 5). The type 1 cells resembled typical Kupffer cells, type 2 cells monocytes, and type 3 and 4 cells the exudate-resident macrophages considered to be a transitional form between exudate and resident macrophages. Type 5 cells may also be a transitional form between the exudate and resident macrophage, or an end-stage macrophage derived from exudate macrophages which have lost their PO activity. Tannic-acid-aldehyde immersion fixation with microwaving may be a useful method in the study of the PO activities of macrophages in biopsied human liver specimens.     

De novo synthesis of d-alanine in germinating Pisum sativum seedlings

The amounts of d-alanine derivatives, γ-l-glutamyl-d-alanine and N-malonyl-d-alanine, increase rapidly during the early growth of pea seeds. Pyruvate-[1?¹⁴C], l-alanine-[U?¹⁴C], d-alanine-[U?¹⁴C], l-alanine-[¹⁵N] and ¹⁵NH₄Cl were therefore fed to the seedlings and the incorporation investigated. Labelling results revealed that pea seedlings can utilize these erogenous compounds to form d-alanine and that labelled l-alanine is effectively converted to the d-enantiomer with retention of ¹⁴C and, largely, ¹⁵N label. Enzyme analyses in vitro provided additional evidence that the extract of pea seedlings catalyzes the direct conversion of l-alanine to d-alanine. The data suggest that the de novo synthesis of d-alanine in pea seedlings occurs by a racemase reaction.     

N-acetyl conjugates of basic amino acids newly identified in rat urine

Ninhydrin-negative conjugates of basic amino acids were isolated from rat urine and were characterized. The following conjugates of basic amino acids are the compounds newly identified in animal urine specimens, N^α-acetyl-N^π-methylhistidine, N^α-(N-acetyl-β-alanyl)histidine (N-acetylcarnosine), N^α-acetyl-N^G,N^′G-dimethylarginine, N^α-acetyl-N^G,N^G-dimethylarginine, and N^α-acetyl-N^?,N^?,N^?-trimethyllysine.     

Mass spectrometric and mutational analyses reveal Lys-6-linked polyubiquitin chains catalyzed by BRCA1-BARD1 ubiquitin ligase

The breast and ovarian cancer suppressor BRCA1 acquires significant ubiquitin ligase activity when bound to BARD1 as a RING heterodimer. Although the activity may well be important for the role of BRCA1 as a tumor suppressor, the biochemical consequence of the activity is not yet known. Here we report that BRCA1-BARD1 catalyzes Lys-6-linked polyubiquitin chain formation. K6R mutation of ubiquitin dramatically reduces the polyubiquitin products mediated by BRCA1-BARD1 in vitro. BRCA1-BARD1 preferentially utilizes ubiquitin with a single Lys residue at Lys-6 or Lys-29 to mediate autoubiquitination of BRCA1 in vivo. Furthermore, mass spectrometry analysis identified the Lys-6-linked branched ubiquitin fragment from the polyubiquitin chain produced by BRCA1-BARD1 using wild type ubiquitin. The BRCA1-BARD1-mediated Lys-6-linked polyubiquitin chains are deubiquitinated by 26 S proteasome in vitro, whereas autoubiquitinated CUL1 through Lys-48-linked polyubiquitin chains is degraded. Proteasome inhibitors do not alter the steady state level of the autoubiquitinated BRCA1 in vivo. Hence, the results indicate that BRCA1-BARD1 mediates novel polyubiquitin chains that may be distinctly edited by 26 S proteasome from conventional Lys-48-linked polyubiquitin chains.     

Improvement of the glutaryl-7-aminocephalosporanic acid acylase activity of a bacterial gamma-glutamyltranspeptidase

7-Aminocephalosporanic acid (7-ACA) is an important material in the production of semisynthetic cephalosporins, which are the best-selling antibiotics worldwide. 7-ACA is produced from cephalosporin C via glutaryl-7-ACA (GL-7-ACA) by a bioconversion process using d-amino acid oxidase and cephalosporin acylase (or GL-7-ACA acylase). Previous studies demonstrated that a single amino acid substitution, D433N, provided GL-7-ACA acylase activity for gamma-glutamyltranspeptidase (GGT) of Escherichia coli K-12. In this study, based on its three-dimensional structure, residues involved in substrate recognition of E. coli GGT were rationally mutagenized, and effective mutations were then combined. A novel screening method, activity staining followed by a GL-7-ACA acylase assay with whole cells, was developed, and it enabled us to obtain mutant enzymes with enhanced GL-7-ACA acylase activity. The best mutant enzyme for catalytic efficiency, with a k(cat)/K(m) value for GL-7-ACA almost 50-fold higher than that of the D433N enzyme, has three amino acid substitutions: D433N, Y444A, and G484A. We also suggest that GGT from Bacillus subtilis 168 can be another source of GL-7-ACA acylase for industrial applications.     

Milk-clotting Enzyme from Microorganisms

The milk-clotting activity of Mucor-rennin obtained from Mucor pusillus Lindt, was not changed by the addition of DFP in the reaction mixture. This finding suggested the probable absence of a serine residue at the active center of the enzyme. Sulfhydryl reagents such as Nekelgon, N-ethyl maleimide, PCMB failed to influence the milk-clotting reaction, indicating that a. reactive sulfhydryl group is not required for the enzymatic activity. The activity was inhibited when Mucor-rennin was treated with iodine at pH higher than 5.0. It was shown that ¹³¹I₂ was incorporated into the enzyme. When Mucor-rennin was photooxidized in the presence of methylene blue, the milk-clotting activity was inactivated. In this case, tyrosine, tryptophan, and histidine residues in the enzyme were oxidized. Among these amino acids, the histidine residue was more rapidly oxidized than other amino acids. A parallel relation was observed between the decrease of the amount of histidine residue and the inactivation of the enzyme. From these results, it is concluded that the histidine residue present in Mucor-rennin has a relation to the active center of this enzyme.