Identification of a peptidase which processes atrial natriuretic factor precursor to its active form with 28 amino acid residues in particulate fractions of rat atrial homogenate

With the objective of identifying specific peptidase responsible for the processing of atrial natriuretic factor precursor pro-ANF to the circulating active form ANF (99-126), a fluorometric assay method was devised using synthetic fluorogenic substrate Boc-Ala-Gly-Pro-Arg-MCA(methylcoumarinamide) which contains the amino acid sequence immediately adjacent to the arginyl peptide bond which is cleaved in the natural processing of pro-ANF. A protease which selectively cleaves this bond and produces the natural circulating peptide was identified in the particulate fraction of rat atrial homogenate and was solubilized by 1.6 M KCl. It was partially purified by affinity chromatography heparin-agarose column and was shown to be a serine protease. Its reaction product with natural pro-ANF was identified as ANF (99-126) containing 28 amino acid residues.     

Formate production from methanol by formaldehyde dismutase coupled with a methanol oxidation system

Summary Formaldehyde dismutase was greatly stabilized by immobilization in a urethane prepolymer (PU-6). The immobilized enzyme exhibited stochiometrical dismutation of formaldehyde to methanol and formate in several repeated reactions. Conversion of methanol to formate occurred in a reaction with an immobilized enzyme system consisting of alcohol oxidase, catalase and formaldehyde dismutase, and with an intact cell-mixture of Hansenula polymorpha and Pseudomonas putida. Furthermore, the stability of the cell-mixture during repeated reactions was greatly improved by the immobilization, the 600 mM methanol added periodically being converted to formate in a 75% yield in 12 h. The immobilized cellsystem was also effective for the conversion of several aliphatic alcohols, C₁ to C₄, to the corresponding acids.     

Isolation of chicken anemia agent with MDCC-MSB1 cells from chickens in the field

An attempt was made to isolate chicken anemia agent (CAA) from chickens suffering from anemia in the field by using MDCC - MSB1 , which was an established cell line derived from Marek's disease lymphoma. When 99 chickens of 15 flocks were examined, CAA was isolated from 58 chickens of 12 flocks. The rate of CAA isolation with MDCC - MSB1 cells was almost the same as that determined by an in vivo method by chick inoculation. It was shown that CAA was more closely concerned with anemic diseases of chickens in the field than fowl adenoviruses.     

The effect of chemicals on the activity of deglycosylated Aureobasidiumβ-fructofuranosidases

The effects of chemicals such as metal ions and typical organic enzyme inhibitors on the activity of deglycosylated β-fructofuranosidases ( P -1 and P -2) from Aureobasidium were observed and compared with those of native enzymes. P -1 and P -2 enzymes became sensitive to metal ions, such as Fe²⁺, Co²⁺, Ni²⁺ and Al³⁺, after deglycosylation. The enzymes were also inhibited by a sulphydryl reagent (monoiodoacetic acid), a peptide-hydrolysing reagent (hydroxylamine) and chelating reagents (sodium citrate, EDTA and sodium azide) after deglycosylation. The importance of deglycosylation for the determination of the true characteristics of the enzymes against chemicals is discussed.     

Immobilization ofβ-fructofuranosidase fromAureobasidium on DEAE-cellulose

Summary -Fructofuranosidase, which produces fructo-oligosaccharides (1-kestose and nystose) from sucrose, was purified fromAureobasidium and immobilized on DEAE-cellulose at especially high efficiency (95%). The enzymatic profiles of the immobilized enzyme were almost identical to those of the native form except that the stability was slightly improved. The immobilized enzyme was stable during long-term continuous reaction for up to 360 h.     

Molecular cloning of the gene which encodes beta-N-acetylglucosaminidase from a marine bacterium, Alteromonas sp. strain O-7.

The gene encoding the periplasmic beta-N-acetylglucosaminidase (GlcNAcase B) from a marine Alteromonas sp. strain, O-7, was cloned and sequenced. The protein sequence of GlcNAcase B revealed a highly significant homology with Vibrio GlcNAcase and alpha- and beta-chains of human beta-hexosaminidase.     

Crystallographic aspects of sub-elementary cellulose fibrils occurring in the wall of rose cells culturedin vitro

Summary Quantities of disencrusted sub-elementary cellulose fibrils from the cell wall of rose cells culturedin vitro were prepared. Following an X-ray and electron diffraction analysis, these fibrils gave a cellulose diffraction pattern which presented only two strong equatorial diffraction spacings at 0.409 and 0.572 nm indicating that the fibrils have a crystalline structure resembling that of cellulose IV_I. This observation is best explained in terms of a lateral disorganization of the cellulose chains within the fibrils. This disorganization cannot be eliminated and is connected with the small width of the fibrils which contain from 12 to 25 cellulose chains only. In these fibrils, most of the cellulose chains are superficial and not locked with neighboring chains in a tight hydrogen bond system as in thicker cellulose microfibrils.     

Electron diffraction from the primary wall of cotton fibers

Summary Electron diffraction patterns have been obtained from selected areas of disencrusted microfibrils isolated from the primary cell wall of cotton fibers. The resultant fiber diagram has the same meridional repeat distance as a corresponding pattern of secondary wall microfibrils but differs markedly in the equatorial reflections. The primary wall diagram displays only two strong equatorial reflections centered at 0.570 nm and 0.416 nm. The similarity of these spacings with those of cellulose IV suggests that the crystalline structure of the primary wall cellulose is similar to that of cellulose IV_I and is best explained in term of native cellulose I crystals having good longitudinal coherence (i.e., coherence along the length of the microfibrils) but with poor lateral organization of the network of inter chain hydrogen bonds. Similar results were also obtained for other primary wall specimens.     

Three-dimensional structure of a highly thermostable enzyme, 3-isopropylmalate dehydrogenase of Thermus thermophilus at 2.2 A resolution.

The three-dimensional structure of the highly thermostable 3-isopropylmalate dehydrogenase (IPMDH) from Thermus thermophilus has been determined by the multiple isomorphous replacement method and refined to 2.2 A resolution. The final R-factor is 0.185 for 20,307 reflections. The crystal asymmetric unit has one subunit consisting of 345 amino acid residues. The polypeptide chain of this subunit is folded into two domains (first and second domains) with parallel alpha/beta motifs. The domains are similar in their conformations and folding topologies, but differ from those of the NAD-binding domains of such well-known enzymes as the alcohol and lactate dehydrogenases. A beta-strand that is a part of the long arm-like polypeptide protruding from the second domain comes into contact with another subunit and contributes to the formation of an isologous dimer with a crystallographic 2-fold symmetry. Close subunit contacts are also present at two alpha-helices in the second domain. These helices strongly interact hydrophobically with the corresponding helices of the other subunit to form a hydrophobic core at the center of the dimer. Two large pockets that exist between the first domain of one subunit and the second domain of the other include the amino acid residues responsible for substrate binding. These results indicate that the dimeric form is essential for the IPMDH to express enzymatic activity and that the close subunit contact at the hydrophobic core is important for the thermal stability of the enzyme.