Phenylalanine ammonia-lyase: Purification and characterization from soybean cell suspension cultures

Soybean cell suspension cultures (Glycine max L. cv. Kanrich) grown on high-nitrogen medium produce 50 mU/g fresh wt of phenylalanine ammonia-lyase [EC 4.1.3.5] 7–9 days after inoculation. Nitrate was not limiting when the peak of enzyme activity was reached. Phenylalanine ammonia-lyase was purified 53-fold to essentially electrophoretic homogeneity from cell extracts with 10% recovery. The enzyme was stable in crude extracts and through most stages of purification. No activity could be detected with tyrosine as substrate in either crude extracts or purified enzyme. The electrophoretic mobility was somewhat less than that of the enzyme from maize but both eluted from an agarose column at the same position and the molecular weight of the subunit was similar for both enzymes. Thus the soybean enzyme is composed of four subunits and the native enzyme is ~330,000 M_r. The variation in structure and/or size and availability of hydrophobic regions among phenylalanine ammonia-lyases from four sources (potato, maize, Rhodotorula glutinis, and soybean) was shown by the different elution patterns they exhibited on columns of ω-aminoalkyl agarose (agarose-C_n-NH₂, n = 0 to 8). The order of increasing hydrophobicity is soybean, potato, maize, R. glutinis. The soybean enzyme exhibited negative cooperativity before hydroxylapatite chromatography and positive cooperativity afterward. This is the first example of positive cooperativity observed for phenylalanine ammonia-lyase.     

XY female mice express H-Y antigen

Karyotypically XY individuals of the C57BL/6J-Y^POS mouse stock develop as females or hermaphrodites, but never as normal males. The aberrant sexual development results from the interaction of the C57BL/6J genetic background with the M. poschiavinus-derived Y chromosome. XY females from this stock were assayed for H-Y antigen. By the criteria of skin-grafting, the cell-mediated lympholysis test, and the popliteal lymph node assay, these XY females are antigenically indistinguishable from normal C57BL/6 males. Implications for the hypothesis that H-Y antigen induces formation of the mammalian testis are discussed.     

Dinitrogen fixation in male-sterile soybeans

Partial male-sterile (ms₄/ms₄) soybeans (Glycine max L. Merr.) and their fertile isoline (Ms₄/Ms₄) were grown in adjoining field plots. From 62 until 92 days after emergence, the nitrogenase activity, assayed by acetylene reduction, of the average male-sterile plant was approximately twice that of the average fertile plant. At approximately 100 days after emergence, the assayable nitrogenase activity of the fertile plants fell to zero, whereas the nitrogenase of the partial male-sterile plants continued to be active for two additional weeks. Thus, this male-sterile plant seems to fix dinitrogen both at a higher rate and over a longer duration than does its fertile isoline.     

Hydroponic growth and the nondestructive assay for dinitrogen fixation

Hydroponic growth medium must be well buffered if it is to support sustained plant growth. Although 1.0 millimolar phosphate is commonly used as a buffer for hydroponic growth media, at that concentration it is generally toxic to a soybean plant that derives its nitrogen solely from dinitrogen fixation. On the other hand, we show that 1.0 to 2.0 millimolar 2-(N-morpholino)ethanesulfonic acid, pK_a 6.1, has excellent buffering capacity, and it neither interferes with nor contributes nutritionally to soybean plant growth. Furthermore, it neither impedes nodulation nor the assay of dinitrogen fixation. Hence, soybean plants grown hydroponically on a medium supplemented with 1.0 to 2.0 millimolar 2-(N-morpholino)ethanesulfonic acid and 0.1 millimolar phosphate achieve an excellent rate of growth and, in the absence of added fixed nitrogen, attain a very high rate of dinitrogen fixation. Combining the concept of hydroponic growth and the sensitive acetylene reduction technique, we have devised a simple, rapid, reproducible assay procedure whereby the rate of dinitrogen fixation by individual plants can be measured throughout the lifetime of those plants. The rate of dinitrogen fixation as measured by the nondestructive acetylene reduction procedure is shown to be approximately equal to the rate of total plant nitrogen accumulation as measured by Kjeldahl analysis. Because of the simplicity of the procedure, one investigator can readily assay 50 plants individually per day.     

Chicken liver fatty acid synthetase dimer: Evidence of asymmetrical structure from iodoacetamide inhibition studies

The condensing component of chicken liver fatty acid synthetase is inhibited by a sulfhydryl reagent, iodoacetamide, with a second-order rate constant of 0.23 M^–1 sec^–1 at pH 7.0 and 0. Complete inactivation requires the modification of approximately 8-SH groups per dimer of the enzyme. Quantitation of the extent of inactivation in the presence of i mM acetyl CoA (which completely protects the enzyme against inactivation) and in its absence shows that complete inactivation results from the binding of approximately 1.1 tool of carboxamidomethyl residues per dimer. These data are consistent with the proposed functional asymmetry of the enzyme.     

Carboxyfluorescein as a probe for liposome-cell interactions effect of impurities,and purification of the dye

Impurities in 5(6)-carboxyfluorescein can affect phospholipid vesicle stability and apparent rates of carboxyfluorescein transfer into cells. Thorough purification and characterization of the dye are thus important to many applications with vesicles and/or cells. The dye can be purified by adsorption chromatography on a hydrophobic gel, following treatment with activated charcoal and precipitation from ethanol-water. The 5- and 6-carboxy-isomers can be separated from each other (though for most purposes it is not necessary to do so) by synthesis, crystallization, and hydrolysis of the diacetate derivatives. Purification is monitored by thin-layer and high pressure chromatography.     

Energetics of mice in stable and unstable social conditions: Evidence of an air-borne factor affecting metabolism

The metabolic rates of laboratory mice were compared in three conditions: isolated mice, mice paired together over six days (stable groups), and mice paired with strange partners daily (unstable groups). Stable pairs had 15% lower metabolic rates than either isolated or unstable pairs. In other experiments when two mice were placed in separate metabolic chambers and connected together via an air flow, the metabolic rate of the recipient in the series was 35% lower than the donor. The data suggest that a ‘factor’ produced by the donor mouse was passed via the air supply into the recipient's chamber.     

Transdetermination of Drosophila imaginal discs cultured in vitro

Imaginal discs of Drosophila melanogaster undergo transdetermination when cultured in vivo in the abdominal cavity of adult female hosts. We report here that leg discs cultured in vitro, in a recently developed system, also undergo transdetermination. Whether cultured in vivo or in vitro, leg discs produce a similar range of specific transdetermined structures. Moreover, in comparison to discs cultured in vivo, the discs cultured in vitro exhibit a similar correlation between the amount of growth and the total frequency of transdetermination.