Hydrogen-ion binding by tobacco-mosaic-virus protein polymers.

Hydrogen ion titration curves of tobacco mosaic virus protein have been measured in various conditions of protein concentration, temperature, ionic strength, and rate of pH change. The polymers present at each stage are deduced from turbidity and sedimentation data, plus published information. A simple semi-quantitative analysis of the curves is given, and the pK values of the two abnormal carboxylates in single helix are estimated as 6.4 and about 7.0. Disks, and some faster-forming unknown polymers in the same size range, have been abnormal carboxylate with pK 6.9. These results are most easily interpreted in terms of electrostatic interactions between carboxylates, probably at the axial ends of the protein subunits.     

Small-angle X-ray-scattering investigation and structural-model study of the fatty-acid synthetase from pig liver (author's transl)

The structure of the fatty acid synthetase from pig liver was studied on models based upon structural and functional properties selected from pertinent results available from numerous investigations carried out with fatty acid synthetases from this and other sources. When comparing small-angle X-ray-scattering curves calculated with these models and curves obtained from small-angle X-ray-scattering experiments carried out with the pig-liver enzyme, we tried to select a model which would lead to an acceptable correlation between the calculated and the experimental curves and at the same time fulfil the known structural and functional requirements. The comparison of the curves was started with a model of low complexity. The observed discrepancy, together with arguments from the structural and the functional properties, helped decide which is the next most reasonable model to be considered. This procedure was repeated for five models of increasing complexity. In the model which led to the best fit the multienzyme complex is composed of two halves in an assymetric conformation including hollow spaces. This highly anisotropic model would imply that the two halves change their conformation each time a synthetic cycle is completed and that the growing fatty acid is handed over from one half to the other.     

Isolation of pure myocardial subcellular organelles.

A procedure is outlined for the isolation of three pure myocardial subcellular fractions by sucrose gradient ultracentrifugation. The purity of the sarcolemmal (SL) and sarcoplasmic reticulum (SR) fragments and mitochondria were documented by marker enzyme assays and SL purity by electronmicroscopy.     

Temperature acclimation in the Mongolian gerbil (Meriones unguiculatus): biochemical and organ weight changes.

1. Adult Mongolian gerbils (Meriones unguiculatus) were acclimated to 5 +/- 1, 24 +/- 1 and 34 +/- 1 degrees C for 6-8 weeks. 2. Body weights of temperature acclimated gerbils did not differ significantly from controls. Organ wt/body wt ratios of liver, kidney and heart increased in cold-acclimated and decreased in heat-acclimated gerbils. Adrenal wt/body wt ratio increased in the cold and was unchanged in the heat. Relative weights of brain, spleen, lungs, brown fat and ovaries + uterus did not change with temperature acclimation. 3. Cold acclimation produced significant increases in specific and total activity of brown fat alpha GPO and liver SO and AAO and in total activity of kidney SO; a significant decrease in liver mitochondrial ADP/O ratio with succinate as substrate; and no change in brown fat SO or liver alpha KGO. 4. Heat acclimation produced significant decreases in specific and total activity of liver and kidney SO, and in total activity of brown fat SO and alpha GPO, and liver AAO and alpha KGO. 5. The combined biochemical and organ wt changes seen in temperature-acclimated gerbils suggest that this species is capable of altering its metabolic thermogenic potential in response to a wide range of ambient temperatures.     

5'-nucleotidase from the electric ray electric lobe. Primary structure and relation to mammalian and procaryotic enzymes.

A cDNA encoding a 5'-nucleotidase was identified by screening a lambda gt10 cDNA library from the electric lobe of Discopyge ommata using a cDNA probe containing the complete open reading frame coding for the rat liver enzyme. Nucleotide sequence analysis defines an open reading frame of 577 amino acids, corresponding to a calculated molecular mass of 63,833 Da. The N-terminus of the mature protein, as determined by direct protein sequencing, is preceded by 29 amino acid residues comprising a signal peptide. The C-terminus contains a stretch of hydrophobic amino acids, considered to be cleaved on post-translational modification and exchanged for glycosylphosphatidylinositol as a membrane anchor. The predicted protein contains four potential N-linked glycosylation sites. Electric ray 5'-nucleotidase shares 61% amino acid identity with the enzymes from rat liver and human placenta, and about 23% with bacterial proteins possessing 5'-nucleotidase activity and also additional enzyme activities like UDP-glucose hydrolase. Polyclonal antibodies raised against 5'-nucleotidase from mammalian sources or the electric ray electric organ reveal mutual cross-reactivity. Interestingly, there are 5-7 domains highly conserved in procaryotes and vertebrates in enzymes exhibiting 5'-nucleotidase, 3'-nucleotidase or phosphodiesterase activity. 5'-nucleotidase isolated from Torpedo electric organ hydrolyzes UDP-glucose at 8% of the rate of AMP hydrolysis. The possible phylogenetic origin of vertebrate 5'-nucleotidase from multifunctional nucleotide hydrolases is discussed.     

Evidence for an apical sorting signal on the ectodomain of human aminopeptidase N.

In polarized epithelial cells aminopeptidase N is targeted to the apical membrane. The aim of this study was to determine whether a sorting signal is necessary for its correct transport to the apical membrane and, if so, to localize this sorting signal to one of the domains of the transmembrane protein. Anchor-minus aminopeptidase N, consisting of the hemagglutinin signal peptide including its cleavage site, and the ectoplasmic domain of human aminopeptidase N were stably expressed in Madin-Darby canine kidney cells cultured on polycarbonate filters. By measurement of the enzymatic activity it was found that the anchor-minus aminopeptidase N was secreted in a polarized manner to the apical side. As a reference the secretion of the secretory granule protein, cystatin C, was likewise studied. Cystatin C was found to be secreted in a nonpolarized manner to both domains. Our data thus show that human aminopeptidase N carries an apical sorting signal and that it is localized on the ectodomain of the enzyme.