Nitrobacter agilis Cytochrome c-550: Isolation, Physicochemical and Enzymatic Properties, and Primary Structure

Nitrobacter agilis cytochrome c-550 was purified to an electrophoreticallyhomogeneous state, and some of its properties were determined.The cytochrome showed an absorption peak at 410 nm in the oxidizedform, and peaks at 416, 521 and 550 nm in the reduced form.Its isoelectric point was 8.1 at 5?C. Analysis of the aminoacid composition showed that the cytochrome molecule was composedof 108 amino acid residues, 16 of which were lysine residues. The cytochrome reacted rapidly with N. agilis cytochrome c oxidaseand yeast cytochrome c peroxidase and more slowly with Pseudomonasaeruginosa nitrite reductase and bovine cytochrome c oxidase.The reactivities with these redox enzymes suggested that thecytochrome might be an evolutionary stage between bacterialand eukaryotic cytochromes c. The primary structure of the cytochrome from the N-terminusto the 85th residue was determined. The N-terminal sequencewas homologous to the corresponding portion of the primary structureof horse cytochrome c. ¹ Present adress: Department of Chemistry, Faculty of Science,Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo,152, Japan. (Received December 3, 1981; Accepted January 28, 1982)     

A cofactor protein required for actin activation of myosin Mg2+ATPase activity in leukemic myeloblasts

The Mg2+ATPase activity of the myosin of a myeloid leukemia cell line (Ml) was not activated by purified Ml actin or by muscle actin alone. Activation required the presence of a cellular fraction as a cofactor in addition to the actin, when Mg2+ATPase was stimulated as much as 20-fold. The cofactor was partially purified and characterized. 1) Its molecular weight was estimated as 45,000 to 55,000 daltons by gel filtration and as 45,000 daltons by SDS polyacrylamide gel electrophoresis. 2) The cofactor was a light chain kinase that phosphorylated both the L1 and L2 light chains of the Ml cell myosin, but not the L3 or heavy chain.     

Clinical analysis on steroids. XII. Occurrence of D-homoannulation during the hot acid hydrolysis of 5β-pregnane-3α,20α-diol disulfate

Two D-homosteroids were isolated from the hydrolyzate of 5β-pregnane -3α,20α-diol disulfate (II) when it was refluxed in 3N hydrochloric acid. The structures of these steroids have been elucidated as 17α-methyl-D-homo-5β-androstane-3α, 17aβ-diol (VI) and 17α-methyl-17aγb-chloro-D-homo-5β-androstan-3α-ol (VIII) by instrumental analyses. The former was identical with a synthetic specimen derived from 5β-pregnane-3α,20β-diol di-sulfate (IV) by uranediol rearrangement. The main hydrolyzates obtained were 17α-ethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol (V) and 5β-pregnane-3α, 20α-diol (III).     

Synthesis of a reactive ATP analog and its preliminary application as an affinity labeling reagent

A reactive ATP analog, N6-(6-bromoacetamidohexyl)-AMP.PCP, was synthesized in an attempt to covalently label the binding sites for adenine nucleotides, especially ATP, of various enzymes which utilize adenine nucleotides as substrates, cofactors, inhibitors or allosteric effectors. This reagent rapidly inactivated rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (GPD), myokinase (MK), and creatine kinase (CK) under very mild conditions. Adenine nucleotide substrates prevented the inactivation. In the case of GPD, complete inactivation was observed when 1 mol of the reagent per mol of enzyme subunit was incorporated into the enzyme. These results indicate that the present ATP analog may be useful as an affinity labeling reagent for various adenine nucleotide-dependent enzymes.     

Interaction of subunits of polypeptide chain elongation factor I from pig liver. Formation of EF-1alpha.EF-1betagamma and EF-1beta complexes

In the preceding papers, we showed that one of the two complementar factors of polypeptide chain elongation factor 1 (EF-1) from pig liver, EF-1alpha, functionally corresponds to bacterial EF-Tu (Nagata, S., Iwasaki, K., and Kaziro, Y. (1976) Arch. Biochem. Biophys. 172, 168), while the other, EF-1betagamma, as well as one of its subunits, EF-1beta, corresponds to bacterial EF-Ts (Motoyoshi, K. and Iwasaki, K. (1977) J. Biochem. 82, 703). Therefore, the interaction between EF-1alpha and EF-1 betagamma or EF-1beta was was examined and the following results were obtained. i) EF-1betagamma catalytically promoted the exchange of [14C]GDP bound to EF-1alpha with exogenous [3H]GDP. ii). In the absence of the exogenous guanine nucleotide, EF-1betagamma as well as EF-1beta could displace GDP bound to EF-1alpha to form an EF-1alpha.EF-1betagamma as well as an EF-1alpha.EF-1beta complex. iii) The occurrence of EF-1alpha.EF-1betagamma and EF-1alpha.EF-1beta complexes was demonstrated by gel filtration on Sephadex G-150. These results strongly indicate that the mechanism of the action of EF-1betagamma or EF-1beta in converting EF-1alpha.GDP into EF-1alpha.GTP is analogous to bacterial EF-Ts, and the reaction is accomplished by the following reactions; EF-1alpha.GDP + EF-1betagamma (or EF-1beta) in equilibrium EF-1alpha.EF-1betagamma (or EF-1beta) + GDP; EF-1alpha.EF-1beta (or EF-1beta) + GTP IN EQUILIBRIUM EF-1alpha.GTP + EF-1betagamma (or EF-1beta).     

Surface charge of protoplasts and their significance in cell-cell interaction

-potential of mesophyll protoplasts of tobacco (Nicotiana tabacum L.), petunia (Petunia hybrida Hort.), turnip (Brassica rapa L.) and cowpea (Vigna unguiculata L. Walpers) was determined by use of a cell electrophoresis apparatus. All protoplasts examined showed a constant negative value of-10 to-35 mV. The addition of CaCl₂ nullified the -potential of tobacco protoplasts. This phenomenon is explained by DLVO theory of colloid science, which has been successfully applied to animal cells. Furthermore, positively charged polymers reversed the -potential to positive values. Treatment of the protoplast surface with several enzymes was carried out to characterize the chemical nature of suface charges. The removal of surface charges was most conspicuous by the treatment of acid phosphatase (EC 3.1.3.2), but did not occur upon treatment with -neuraminidase (EC 3.2.1.18) or Streptomyces griseus pronase. Thus a major part of the surface charge originates from the phosphate groups at the cell membrane. The significance of these studies for the properties of the protoplast surface in cell adhesion is discussed.     

Changes in actin-related gelation of crude cell extracts during differentiation of myeloid leukemia cells

Gelation of extracts of a myeloid leukemia cell line (Ml) was compared before and after differentiation induced with conditioned medium (CM) from rat embryo cells. Although an extract of Mml cells, a macrophage line derived from Ml line, gelled when warmed in the presence of 2 mM MgCl2, undifferentiated Ml cells gelled only after dialysis and a supplement of exogenous actin. After differentiation had been induced, an addition of exogenous actin, but not dialysis, was needed for gelation. Small amounts of KCl always inhibited the gelation of the control Ml cell extracts, but they promoted gelation of the CM-treated Ml and Mml cell extracts. Thus, the dialysis required for gelation of the control Ml cell extract appears to be necessary for the exclusion of endogenous KCl. Several possible mechanisms for the KCl control of gelation, as well as different requirements of exogenous actin needed for gelation are discussed based on the results of our experiments.     

Enhancement by transforming growth factor-beta 1 (TGF-beta 1) of the proliferation of leukemic blast progenitors stimulated with IL-3.

We studied the effect of transforming growth factor-beta 1 (TGF-beta 1) on colony formation of leukemic blast progenitors from ten acute myeloblastic leukemia (AML) patients stimulated with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), interleukin-6 (IL-6), or interleukin-1 beta (IL-1 beta). These CSFs and interleukins by themselves stimulated the proliferation of leukemic blast progenitors without adding TGF-beta 1. G-CSF, GM-CSF, and IL-3 stimulated blast colony formation in nine patients, IL-6 stimulated it in five, and IL-1 beta stimulated in four. TGF-beta 1 significantly reduced blast colony formation stimulated by G-CSF, GM-CSF, or IL-6 in all patients. In contrast, TGF-beta 1 enhanced the stimulatory effect of IL-3 on blast progenitors from three cases, while in the other seven patients TGF-beta 1 reduced blast colony formation in the presence of IL-3. To study the mechanism by which TGF-beta 1 enhanced the stimulatory effect of IL-3 on blast progenitors, we carried out the following experiments in the three patients in which it occurred. First, the media conditioned by leukemic cells in the presence of TGF-beta 1 stimulated the growth of leukemic blast progenitors, but such effect was completely abolished by anti-IL-1 beta antibody. Second, the addition of IL-1 beta in the culture significantly enhanced the growth of blast progenitors stimulated with IL-3. Third, leukemic cells of the two patients studied were revealed to secrete IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) constitutively; the production by leukemic cells of IL-1 beta and TNF-alpha was significantly promoted by TGF-beta 1. Furthermore, the growth enhancing effect of TGF-beta 1 in the presence of IL-3 was fully neutralized by anti-IL-1 beta antibody. These findings suggest that TGF-beta 1 stimulated the growth of blast progenitors through the production and secretion of IL-1 beta by leukemic cells.     

Purification and properties of human placental aminopeptidase B.

Aminopeptidase B (EC 3.4.11.6; L-arginyl-beta-naphthylamidase) was purified 1,800-fold from human placental cytoplasm and characterized. The enzyme was subjected to ammonium sulfate fractionation and a series of chromatographies on DE-52, hydroxylapatite, Bio-gel A 0.5 m and L-arginine-Sepharose. The native molecular mass of the enzyme was estimated to be 220,000 by gel filtration. The molecular mass was estimated to be about 83,000 by SDS/PAGE in the absence of 2-mercaptoethanol, suggesting that the enzyme exists in a polymeric form. The isoelectric point of the enzyme was 5.4. The purified enzyme was most active at pH 7.2 with L-arginyl-beta-naphthylamide as substrate and the Km value for this enzyme was 0.3 mmol/l. Human placental aminopeptidase B was markedly activity by Cl-. Bestatin and arphamenin, low molecular weight peptides, showed appreciable inhibition of this enzyme. However, amastatin and puromycin did not inhibit the enzyme. Bacitracin markedly activated this enzyme.