Characterization of recombinant human renin: Kinetics,pH-stability, and peptidomimetic inhibitor binding

The kinetic behavior andpH-stability of recombinant human renin was analyzed using a new fluorogenic substrate based on the normal P₆-P₃ renin cleavage sequence in human angiotensinogen. The design of this fluorogenic substrate makes possible, for the first time, direct monitoring of the kinetics of proteolytic conversion of prorenin to renin. ThepH-stability profile for renin, measured with the substrate at 25°C, indicated a broad plateau of stability betweenpH 6.0 and 10.0. Analysis of thepH-activity profile of renin for the substrate indicated a minimumK _m (1.8 µM) atpH 7.4 and a maximumV _m betweenpH 7.4 and 8.0. The thermodynamics of the binding of a novel, soluble, peptidomimetic inhibitor to renin indicated it is possible to retain the tight-binding characteristics and enthalpy contributions to binding of larger peptide-derived inhibitors, while reducing inhibitor size and entropic contributions to binding. A novel derivative of the fluorogenic substrate, containing a 3-methyl histidine substitution at the P₂ site, was used to test the recent hypothesis that renin functions by virtue of substrate-directed catalysis.     

Duplications of the X chromosome in males: evidence that most parts of the X chromosome can be active in two copies

Summary We have analysed two duplications of the X chromosome in male patients using chromosome replication and DNA methylation patterns as determinants of the functional status of the duplicated segments. In both cases, the large duplicated regions, Xq12-q22 and Xq26.3-qter, were not inactivated. A review of previously reported male cases revealed that these duplications were also not subject to inactivation. Taken together, the examined duplications cover almost the entire X chromosome except the pericentromeric region and Xq25–26. Thus, most regions of the X chromosome can be present in two functional copies without lethal consequences.     

The duplication in Charcot-Marie-Tooth disease type 1a spans at least 1100 kb on chromosome 17p11.2

Summary Recently, it has been shown that Charcot-Marie-Tooth disease type 1a (CMT1a) is linked with a duplication of a DNA segment that is detected by probe VAW409R3, and that is located on chromosome 17p11.2. Here, we show that this duplication also contains VAW412R3a, but not A10-41 and EW503. Accounting for the duplication in recombination analysis, we found recombinants between CMT1a and EW301 and EW502, but not with A10-41, VAW409R3, and VAW412R3. Using pulsed-field gel electrophoresis analysis, we estimated the minimal size of the duplicated region in CMT1a patients to be 1100 kb.     

Cystatin domains in alpha-2-HS-glycoprotein and fetuin

We have found that chain A of alpha-2-HS-glycoprotein contains two cystatin domains that show closest similarity to those of kininogen. Most likely, the two proteins diverged after the primary duplication of a single cystatin domain as the two cystatin domains of alpha-2-HS-glycoprotein are more similar, especially in disulfide bonding, to the corresponding domains of kininogen than to each other. We also propose that the carboxyl-terminal (non-cystatin) parts of kininogen and alpha-2-HS-glycoprotein contain homologous segments. We suggest that alpha-2-HS-glycoprotein may act as an inhibitor of the cysteine proteinases responsible for bone resorption. We have also found that fetuin is closely related to alpha-2-HS-glycoprotein.     

Paramolybdate anion-exchange resin, an improved catalyst for the C-1-C-2 rearrangement and 2-epimerization of aldoses

Aqueous solutions of molybdate at 90 degrees bring about the inversion of the C-1-C-2 fragment of aldoses having four or more carbon atoms, generating thermodynamically equilibrated mixtures of the starting aldose and its 2-epimer. In some cases, notably with the aldopentoses, substantial proportions of the 3-epimers are produced, as well as 2-epimers that have not undergone inversion of the C-1-C-2 fragment. These side-reactions can be controlled by using the paramolybdate form of an anion-exchange resin (AG MP-1) together with the formate form of the same resin. The latter acts to scavenge unbound molybdate and paramolybdate anions that appear to be responsible for the side reactions.     

The localisation of an Mr 74,000 major chromatin antigen on native salivary chromosomes of Drosophila melanogaster

An antigen making a major contribution to the immune response to Drosophila melanogaster chromatin resides primarily on a nonhistone charge-class family of proteins of Mr 74,000. Immunofluorescence detects this antigen at interbands, puffs and diffuse bands of D. melanogaster salivary chromosomes isolated without exposure to acid fixatives, and on nucleoplasmic ribonucleoprotein droplets. In the electron microscope, gold labelling reveals the binding of monoclonal antibodies specific for the antigen at chromosomal loci generally bearing putative ribonucleoprotein (RNP) particles. However, the locus 3C 11–12 is remarkable in that it bears putative RNP particles but is virtually unlabelled, suggesting protein specificity at different active loci.     

Expression of polyoma virus middle-T antigen in Saccharomyces cerevisiae

The polyoma middle-T gene, lacking its intron, was inserted into a yeast expression plasmid containing the phosphoglycerate kinase promoter. Such plasmids transformed yeast at low frequency and these transformants expressed middle-T antigen at a level of approximately 0.1% cell protein. Furthermore, expression of this protein was frequently lost during growth in liquid culture and this loss of middle-T was accompanied by a twofold increase in the rate of growth. The spontaneous production of a truncated middle-T antigen, lacking the C terminus, was also observed; the expression of this protein did not inhibit the growth rate of the cells. Recovery and analysis of the expression plasmids encoding the truncated molecule showed that a single C X G base pair had been deleted from a run of nine consecutive C X G base pairs (Pyr nucleotide 1239--1247) within the middle-T coding region. This frame-shift mutation results in premature termination of the protein and loss of the strongly hydrophobic region of the molecule believed to be responsible for the membrane association of middle-T antigen.     

Relative Virulence of Meloidogyne incognita Host Races on Soybean

Sensitivity and host efficiency of susceptible (''Lee 68'', ''Coker 156'') and resistant (''Bragg'', ''Centennial'', ''Forrest'', ''Lee 74'') soybean (Glycine max (L.) Merr.) cultivars for races of Meloidogyne incognita (Mi) were determined in greenhouse experiments. Eight Mi populations collected from the southeastern United States were utilized. All Mi races reproduced readily on Lee 68 and Lee 74 and moderately on Forrest and Bragg. Coker 156 exhibited resistance to races 1 and 2, and some race 3 populations, but was very susceptible to certain race 3 and 4 populations. Reproduction of all races was lowest on Centennial. Forrest and Centennial shoot growth was not significantly suppressed by any race. There were no distinct differences in virulence between races except for a race 3 population which reproduced readily on all cultivars, stunting their growth. Considerable variation in reproduction existed within races 1 and 3.     

Adenine nucleotide translocase-dependent anion transport in pea chloroplasts

Pea chloroplasts were found to take up actively ATP and ADP and exchange the external nucleotides for internal ones. Using carrier-free [¹⁴C]ATP, the rate of nucleotide transport in chloroplasts prepared from 12–14-day-old plants was calculated to be 330 μmol ATP/g chlorophyll/min, and the transport was not affected by light or temperature between 4 and 22°C. Adenine nucleotide uptake was inhibited only slightly by carboxyatractylate, whereas bongkrekic acid was nearly as effective an inhibitor of the translocator in pea chloroplasts as it was in mammalian mitochondria. There was no counter-transport of adenine nucleotides with substrates carried on the phosphate translocator including inorganic phosphate, 3-phosphoglycerate and dihydroxyacetone phosphate. However, internal or external phosphoenolpyruvate, normally considered to be transported on the phosphate carrier in chloroplasts, was able to exchange readily with adenine nucleotides. Furthermore, inorganic pyrophosphate which is not transported by the phosphate carrier initiated efflux of phosphoenolpyruvate as well as ATP from the chloroplast. These findings illustrate some interesting similarities as well as differences between the various plant phosphate and nucleotide transport systems which may relate to their role in photosynthesis.     

Dysgenesis-Induced Instability of Rosy Locus Transformation in DROSOPHILA MELANOGASTER: Analysis of Excision Events and the Selective Recovery of Control Element Deletions

Utilizing the method of P-M hybrid dysgenesis-mediated gene transfer to insert rosy locus DNA into various chromosomal locations, we recovered a transformed strain that carries an ry+ transposon inserted in or near the scalloped locus in polytene section 13F on the X chromosome. The resultant product, when stabilized, behaves as a homozygous and hemizygous viable and fertile extreme scalloped allele associated with wild-type expression of the rosy locus. We have labeled this allele, sdry+. This allele has been destabilized by subsequent P-M hybrid dysgenesis, and mutations were recovered that exhibit alterations in the rosy and/or scalloped phenotypes. Representative samples of all phenotypic classes have been characterized by Southern blot analyses of restricted DNA. The most common events are excisions of DNA wholly internal to the transposon and representing sections of rosy DNA. In addition to loss of rosy locus function, such excisions affect the scalloped locus expression.--A second dysgenesis experiment was carried out involving an ry+ transposon inserted in polytene section 16D on the X chromosome. A minimal estimate of the relative frequency of imprecise excisions, determined in this experiment is 75%.--A successful pilot experiment is described that utilizes dysgenic perturbation of the sdry+ allele to select for small deletions of the 5' noncoding region of the rosy locus.