The reaction product of peptidylglycine alpha-amidating enzyme is a hydroxyl derivative at alpha-carbon of the carboxyl-terminal glycine

The peptidylglycine alpha-amidating enzyme catalyzes a reaction that transforms a carboxyl-terminal glycine-extended precursor into a carboxyl-terminal alpha-amidated peptide. We purified an alpha-amidating enzyme from equine serum by simplified steps including substrate affinity chromatography. With the purified enzyme, we detected an intermediate of the alpha-amidating reaction by high performance liquid chromatography analysis. The production of the intermediate required copper, oxygen, and ascorbate and increased linearly with incubation time. The structure of the intermediate was determined to be a hydroxyl derivative at the carboxyl-terminal glycine by fast atom bombardment mass spectrometry and by proton NMR. The intermediate was readily converted into an alpha-amidated product in alkaline conditions in a nonenzymic fashion. The nonenzymic conversion required no cofactor but was extremely accelerated by the addition of copper ion or at higher temperature. Our data suggest that the direct product of the alpha-amidating reaction is not an alpha-amidated peptide but a hydroxyl derivative at the alpha-carbon of the carboxyl-terminal glycine.     

Mapping of insertion element IS30 in the Escherichia coli K12 chromosome

Summary We identified seven phage clones containing the insertion element IS30 in a phage library mini-set, which includes 476 clones carrying chromosomal segments that cover almost the entire chromosome ofEscherichia coli K12 W3110 (Kohara et al. 1987). We could assign locations and orientations to four copies of IS30 (namedis30A tois30D) on the W3110 chromosome by restriction analysis of phage DNAs containing them. These IS30s were present at the same locations in chromosomes of both W3110 and anotherE. coli K12 strain JE5519, and thus are assumed to be present in otherE. coli K12 derivatives, including early isolates. Among the IS30 copies found, one (is30B) contained a large deletion and possessed only a 181 by stretch of the right terminal region of IS30.EMBL Accession Number: The EMBL accession number of the sequence reported in this paper is X17345     

Effects of Auxin and Anoxia on the Cell Wall Yield Threshold Determined by Negative Pressure Jumps in Segments of Cowpea Hypocotyl

The growth of any plant is based on the enlargement of its constituentcells, which is driven by the uptake of water and is supportedby the irreversible yielding of the cell wall. The latter processis driven by the effective turgor (Pⁱ–Y), i.e., the excessturgor pressure beyond a critical yield threshold (Y), and itis limited by the physiological extensibility () of the cellwall, which serves as the coefficient of proportionality. Ithas been suggested that the plant growth hormone auxin increases, with a resultant promotion of growth, while it does not affectY (Cleland 1977). Any recorded change in Pⁱ has been very small,if any, during the promotion of growth by auxin. We have developed a novel method called the "negative pressurejump" technique which enables us directly and very quickly todetermine in vivo the adjustable yield threshold (Y') with minimumperturbation of the elongation of the cell. The cell turgoris reduced to Y' not osmotically but hydraulically by applicationof tension to xylem vessels from the drain of the xylem perfusionsystem. In the case of segments of hypocotyls of cowpea seedlings,auxin was found to reduced Y' definitively (by 40–80 kPa).The contribution of this decrease is comparable in magnitudeto that of the increase in with respect to the promotion ofgrowth. Once it has been decreased, Y' is not affected by anoxia. (Received March 26, 1990; Accepted May 24, 1990)     

Adduct formation of pyrroloquinoline quinone and amino acid

When pyrroloquinoline quinone (PQQ) is mixed with an amino acid, a corresponding Schiff base PQQ adduct is readily formed between carbonyl groups of PQQ and the primary amino group. A potent growth stimulating effect for microorganisms was observed with the PQQ adduct when it was administered in a culture medium. Although PQQ itself shows a marked growth stimulating effect, PQQ adducts appeared to be more active than authentic PQQ when compared on a molar basis. Conversely, unlike authentic PQQ, PQQ adducts were shown to be less active (greater than or equal to 100-fold) as the prosthetic group for a quinoprotein apo-glucose dehydrogenase when examined by holoenzyme formation by exogenous addition of PQQ or PQQ adducts. These observations suggested that PQQ adduct formation readily occurs during isolation procedures for PQQ from biological materials or PQQ - chromophore from quinoproteins. Therefore, the presence of such adducts gives a PQQ estimation much lower than theoretically expected. As an example, formation, isolation and characterization of PQQ - serine are described.     

Effects of Osmotic Stress, Salt Stress and IAA on the Regeneration of the Resting Membrane Potential after Excision of a Segment from an Intact Plant

Effects of osmotic stress, salt stress and IAA on the regenerationprocess of the transmembrane potential across the xylem/symplastinterface (V_px) of newly excised hypocotyl segments of Vignaseedlings were examined by means of the xylem perfusion method.It took about 8 h under ordinary conditions for an excised segmentto regenerate a membrane potential comparable with that of anintact seedling. Osmotic stress imposed by perfusion of 100-200mM sorbitol solution seemed to accelerate this process. Thiseffect diminished with the removal of sorbitol from the perfusionsolution. The increase in the negativity of V_px in this regenerationprocess resulted from the increases in both passive (V_px inN₂) and respiration-dependent components (V_px). NaCl (50–100mM) did not accelerate the regeneration of the total membranepotential, but significantly promoted an increase in V_px, i.e.electrogenic xylem pump activity. Perfusion of KC1 (50–100mM) or IAA (10^–4M) shortened the regeneration phase upto 2–3 h. The increase in total V_px under salt stressor IAA mainly resulted from the increase of V_px. The effectof those agents on the V_px is discussed, as is the questionof whether the increase in PD after excision should be interpretedas a recovery or regeneration from injury by excision, or asa so-called release from hormonal control. (Received September 3, 1987; Accepted March 14, 1988)     

Dose-response relationship for translocation induction in spermatogonia of the crab-eating monkey (Macaca fascicularis) by chronic gamma-ray-irradiation

The induction of reciprocal translocations in spermatogonia of the crab-eating monkey (Macaca fascicularis) by chronic gamma-irradiation (1.8 x 10(-5) Gy/min, about 0.024 Gy/22 h/day) was examined. The frequencies of translocation per cell were 0.15% at 0.3 Gy, 0.27% at 1.0 Gy and 0.33% at 1.5 Gy. The dose-response relationship for translocation yield was a linear one with a regression coefficient (b) of 0.16 x 10(-2). When the slope (b) of the regression line was compared with that at a high dose rate (0.25 Gy/min, b = 1.79 x 10(-2), it was clear that the induction rate of translocations after chronic gamma-irradiation was only about one-tenth of that after high-dose-rate irradiation. Thus, there was evidence for a pronounced dose-rate effect in the crab-eating monkey.     

Comparative androecium morphogenesis ofSicyos angulatus andSechium edule (Cucurbitaceae)

“Androecium” ofSicyos angulatus andSechium edule is unique in having a solid central column below a head portion with thecae. Its morphogenesis was examined for the two species. The developmental course is composed of two distinct successive phases; (1) establishment of stamen primordia and (2) uplift of the stamen primordia caused by development of a central column below them. In the first phase, there is a difference between the two species; inSicyos angulatus, two bithecal and one monothecal stamen primordia are formed by congenital fusion among preformed five protrsions, whilst inSechium edule, three or four monothecal stamen primordia are formed without fusion. The central column is later produced by intercalary growth in a region below the stamen primordia in both species. Concomitant with central column development, the center of the floral primordium, which was surrounded by the early formed stamen primordia, is raised up to the top of the central column. The central column could be interpreted as a receptacular column, and not as congenitally fused stamen filaments, as currently believed. The “androecium” of the both species is considered an androecium complex, which consists of the stamens and a receptacular column.     

Studies on the function mechanism of a Formosan grey mullet protamine-mugiline beta M6: interaction of the M6 and M6 fragments with DNA.

The interaction of three peptide segments of one component of Formosan grey mullet protamine (mugiline beta M6), obtained by chemical and enzymatic cleavage, with DNA was studied by spectroscopic measurement, thermal denaturation and circular dichroism. The data obtained were then compared with those of whole M6 and other fish protamines such as salmine of salmon and clupeine of herring. M6-B-I, which lacks C-terminal 11 amino acids in M6, showed significantly different properties. It showed remarkably high DNA aggregating ability which was due to a conformational change of DNA from B to A form. The conformational change of DNA induced by the binding of M6-B-I was reproduced by the carboxypeptidase B digestion of DNA-M6 complex. From these results, the arginine-rich, C-terminal domain of the M6 molecule was estimated to be essential for natural DNA binding.     

Thyroid microsomal antigen in Graves' thyroid is not different from that in normal thyroid.

Differences from normal in microsomal antigen (M-Ag) may be involved in the development of autoimmune thyroid disease. We compared the M-Ag in Graves' thyroid immunologically and biochemically to that in normal thyroid. The concentration of M-Ag, measured with an enzyme-linked immunosorbent assay, was significantly greater in the Graves' microsomes than in normal microsomes. Binding of a patient's microsomal antibody to Graves' microsomes was completely inhibited when the serum was first incubated with normal thyroid microsomes. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and Western blotting were done with a monoclonal antibody to denatured M-Ag. In both Graves' and normal thyroids, M-Ag existed as 107-, 101-, and 95-kDa peptides. After incubation with V8 protease, the residual antigenic peptide had a molecular weight of less than 60,000 and after incubation with trypsin, 95- and 87-kDa peptides and several smaller antigenic peptides were found. There were no significant differences in the pattern of normal and Graves' microsomes after digestion. Two-dimensional gel electrophoresis of Graves' microsomes showed that the isoelectric point for the 107-kDa peptide was at pH 7.2; that for the 101-kDa peptide was at pH 6.2, and that for the 95-kDa peptide was at 6.5. These values were not different from those observed for normal microsomes. These results indicate that M-Ag in Graves' thyroid does not differ from that in normal thyroid, and that microsomal antibodies in autoimmune thyroid disease probably do no arise from differences in the antigen.