Purification and properties of cathepsin D from the mammary glands of lactating rabbits

Cathepsin D was purified from the lactating rabbit mammary gland by a rapid procedure, which included fractionation with (NH4)2SO4, acid precipitation, double affinity chromatography on pepstatin-Sepharose 4B and gel filtration on Sephadex G-100, resulting in approximately 360-fold purification of the enzyme over the homogenate and approximately 16% recovery. After isoelectric focusing, the enzyme dissociated into four (pI 5.8, 6.3, 6.5 and 7.2) multiple forms, but appeared homogeneous on polyacrylamide gel electrophoresis. Cathepsin D has a Mr of 45 kDa as determined by Sephadex G-100 column chromatography. On sodium dodecylsulfate/polyacrylamide gel electrophoresis the enzyme gave a single protein band, corresponding to Mr of 45 kDa. The amino acid composition of the enzyme is similar to that of cathepsins D from other tissues. A single N-terminal amino acid was glycine. Cathepsin D contains 6.4% carbohydrates consisting of mannose, galactose, fucose and glucosamine at a ratio of 3:9:2:2. Cathepsin D is inhibited by pepstatin with Ki of 2.5 X 10(-9) M and irreversibly by N-diazoacetyl-N'-2.4-dinitrophenyl-ethylene diamine. The enzyme hydrolyzes bovine hemoglobin with the maximal activity at pH 3.0 with Km = 10(-5) M and HLeu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe with Km = 4 X 10(-5) M and Rcat = 0.95 s-1. The major cleavage sites were Leu15-Tyr16, Phe24-Phe25 and Phe25-Tyr26 during hydrolysis of the oxidized insulin B-chain by cathepsin D.     

Human Long-Latency Auditory Evoked Potentials during Radial Motion of the Sound Source

Human long-latency auditory evoked potentials were studied during simulation with variable-amplitude pulse sequences from a sound source moving to and from the subject. The N1 peak parameters were shown to depend on an accurate estimate of the direction of the change in the distance to the sound source. Differences in the processing of signals that simulated the approaching and/or distancing of the sound source were found in the N1 and P2 component parameters of on- and off-responses as was a more pronounced long negative potential shift in the evoked response to the approaching source as compared to the distancing source.     

Identification of a cDNA/Protein Leading to an Increased P i -uptake in Xenopus laevis Oocytes

In a previous report we documented an increased Na⁺-dependent transport of inorganic phosphate (P _i ) in Xenopus laevis oocytes injected with mRNA isolated from rabbit duodenum (Yagci et al., Pfluegers Arch. 422:211–216, 1992; ref 24). In the present study we have used expression cloning in oocytes to search for the cDNA/mRNA involved in this effect. The identified cDNA (provisionally named PiUS; for P _i -uptake stimulator) lead to a 3-4-fold stimulation of Na⁺-dependent P _i -uptake (10ng cRNA injected, 3–5 days of expression). Na⁺-independent uptake of P _i was also affected but transport of sulphate and l-arginine (in the presence or absence of sodium) remained unchanged. The apparent K _m -values for the induced Na⁺-dependent uptake were 0.26 ± 0.04 mm for P _i and 14.8 ± 3.0 mm for Na⁺. The 1796 bp cDNA codes for a protein of 425 amino acids. Hydropathy analysis suggests a lack of transmembrane segments. In vitro translation resulted in a protein of 60 kDa and provided no evidence of glycosylation. In Northern blots a mRNA of ∼2 kb was recognized in various tissues including different intestinal segments, kidney cortex, kidney medulla, liver and heart. Homology searches showed no similarity to proteins involved in membrane transport and its control. In conclusion, we have cloned from a rabbit small intestinal cDNA library a novel cDNA encoding a protein stimulating P _i -uptake into Xenopus laevis oocytes, but which is not a P _i -transporter itself. Received: 31 July 1996/Revised: 16 October 1996     

Lytic enzymes of Trichoderma and their role in protecting plants from fungal diseases

Lytic enzymes of mycoparasitic fungi of the genus Trichoderma, capable of suppressing several fungal phytopathogens that originate in air or soil, are reviewed. The topics analyzed include (1) regulation of production of chitinases, beta-1,3-glucanases, and proteases; (2) molecular and catalytic properties of purified enzymes; and (3) their in vitro ability to degrade cell walls and inhibit sporulation or germ-tube elongation in various phytopathogenic fungi. Among the results summarized are reports of cloning the expression of genes coding for certain lytic enzymes of Trichoderma spp. These genes are used for obtaining plant transgenes with increased resistance to fungal diseases and Trichoderma transformants that produce higher levels of one lytic enzyme (a chitinase or protease) and thereby exhibit a more pronounced ability to suppress phytopathogenic fungi.     

Low Si combined with drought causes reduced transpiration in sorghum Lsi1 mutant

Plant and Soil - High and stable plant productivity is a major aim in agricultural research. Silicon fertilization improves yields of various crops under stress. Nonetheless, broad application of...