Limited proteolysis patterns of the B chain of insulin.

The oxidized B chain of insulin was used as a simple model for further consideration of limited proteolysis with low substrate:enzyme ratios. With low B chain:trypsin ratios, the ordinarily slower cleavage rate of the -Lys₂₉-Ala₃₀ bond essentially equaled the cleavage saturation rate of the -Arg₂₂-Gly₂₃ bond. This led to the disappearance of octapeptide which ordinarily forms most rapidly. Heptapeptide and alanine, formed mainly by cleavage of the octapeptide, decreased somewhat at high enzyme relative levels. Trypsin added to B chain formed a single chromatographic peak.     

Post-translational heterogeneity of the human vitamin D-binding protein (group-specific component)

The vitamin D-binding protein in human serum (the group-specific component) is an alpha 2-globulin which is genetically polymorphic in all populations studied. Previous work (J. Svasti and B. H. Bowman (1978) J. Biol. Chem. 253, 5188-5194, and J. Svasti, A. Kurosky, A. Bennett, and B. H. Bowman (1979) Biochemistry 18, 1611-1617) has shown that the electrophoretic variations of the proteins controlled by two allelic genes, Gc1 and Gc2, are due to at least three amino acid substitutions between Gc1 and Gc2 (Svasti et al. (1979] and to heterogeneity in the Gc1 phenotype arising from carbohydrate dissimilarities. Gc1 migrates electrophoretically as two protein bands, while Gc2 migrates cathodally as a single band. This study demonstrates a post-translational glycosylation difference occurring in a single area of the Gc1 sequence which accounts for the heterogeneity observed previously. The glycosylation site, a threonine residue, appears to be in a sequence which differs between Gc1 and Gc2. The O-glycosidic bond, which is typical of mucins, is rare in plasma proteins. The cyanogen bromide fragment containing the galactosamine-containing carbohydrate in Gc1 was partially sequenced through 20 residues from the amino terminus. No detectable galactosamine could be found in the homologous cyanogen bromide fragment in Gc2. A new purification procedure for the vitamin D-binding protein in human plasma has been developed. Three chromatographic steps provide purified protein.     

Isomeric lipid signatures reveal compartmentalized fatty acid metabolism in cancer

The cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular FAs and their de novo synthesis. Given that oxidation of de novo synthesized FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates; however, hitherto, all FAs have been considered part of a common pool. To probe potential metabolic partitioning of cellular FAs, cancer cells were supplemented with stable isotope-labeled FAs. Structural analysis of the resulting glycerophospholipids revealed that labeled FAs from uptake were largely incorporated to canonical (sn-) positions on the glycerol backbone. Surprisingly, labeled FA uptake also disrupted canonical isomer patterns of the unlabeled lipidome and induced repartitioning of n-3 and n-6 PUFAs into glycerophospholipid classes. These structural changes support the existence of differences in the metabolic fates of FAs derived from uptake or de novo sources and demonstrate unique signaling and remodeling behaviors usually hidden from conventional lipidomics.     

EPR properties of mixed-valent μ-oxo and μ-hydroxo dinuclear iron complexes produced by radiolytic reduction at 77 K

 Radiolytic reduction at 77 K of oxo-/hydroxo-bridged dinuclear iron(III) complexes in frozen solutions forms kinetically stabilized, mixed-valent species in high yields that model the mixed-valent sites of non-heme, diiron proteins. The mixed-valent species trapped at 77 K retain ligation geometry similar to the initial diferric clusters. The shapes of the mixed-valent EPR signals depend strongly on the bridging ligands. Spectra of the Fe(II)OFe(III) species reveal an S=1/2 ground state with small g-anisotropy as characterized by the uniaxial component (g _z –g _av /2<0.03) observable at temperatures as high as ∼100 K. In contrast, hydroxo-bridged mixed-valent species are characterized by large g-anisotropy (g _z –g _av /2>0.03) and are observable only below 30 K. Annealing at higher temperatures causes structural relaxation and changes in the EPR characteristics. EPR spectral properties allow the oxo- and hydroxo-bridged, mixed-valent diiron centers to be distinguished from each other and can help characterize the structure of mixed-valent centers in proteins. Received: 27 June 1998 / Accepted: 25 February 1999     

The effects of immersion and exercise on prolactin during pregnancy

Prolactin is an important hormone during pregnancy, affecting mother, fetus, and amniotic fluid volume. Immersion is known to affect prolactin levels significantly. To determine the effect of immersion and exercise on the prolactin response during pregnancy, we examined serum prolactin levels at 15, 25, and 35 weeks' gestation and 10 weeks post partum. Twelve women completed 20 min land rest, 20 min immersion in 30 degrees C water to the xiphoid, and 20 min exercise in the water at 60% VO2max. Resting prolactin levels were 1.91 +/- 0.32, 4.55 +/- 0.5, and 5.85 +/- 0.27 nmol.l-1 +/- standard error of the mean at 15, 25, and 35 weeks' gestation, respectively. Postpartum lactating women had a resting mean prolactin level of 3.95 +/- 1.6 versus 0.22 +/- 0.4 nmol.l-1 in non-lactating women. Prolactin levels declined significantly during immersion even after correction for dilution by plasma volume shifts. The immersion response was inversely related to the duration of pregnancy with 29%, 22%, and 12% drops during 15-, 25- and 35-week trials, respectively. Compared to rest, exercise prolactin levels remained depressed during the 15th and 25th week trials. We hypothesize that immersion in water caused prolactin levels to decline.     

Mutations that affect circadian rhythms in Neurospora crassa can alter the reduction of cytochromes by blue light

We have examined membrane fractions from mutant strains of Neurospora crassa that have altered responses to blue light or have altered circadian rhythms. Using an in vitro assay, we assessed whether the mutations affected the levels of photoreducible cytochromes. Three of the mutant strains, prd-1, rib-1, and wc-1, were not qualitatively different from the wild type. The poky strain was found to have high concentrations of photoreducible cytochrome c. After removal of this cytochrome, however, the photoreducible cytochromes in the plasma membrane and endoplasmic reticulum were also similar to those of the wild type. The most significant differences were found in strains mutated at the frq locus, which affects circadian rhythms. In the frq-9 strain, the cytochrome in the endoplasmic reticulum was not detectably reduced by blue light. The frq-1 mutation caused a significant shift in the spectrum of blue-light-reduced cytochrome in the endoplasmic reticulum.     

On the dependence of the elasticity and strength of cancellous bone on apparent density

This paper presents a statistical analysis of the pooled data from a number of previous experiments concerning the dependence of the Young's moduli and strength of cancellous bone tissue upon apparent density. The results show that both the Young's moduli and the strength are proportional to the square of apparent density of the tissue and are therefore proportional to one another. It is shown that the coefficient of proportionality is different for human and bovine tissue. It is concluded that the suggestion of Wolff (Das Gesetz der Transformation der Knochen, Hirschwald, Berlin, 1892) that compact bone tissue is simply more dense cancellous bone tissue is not an accurate statement when only the mechanical properties of these two tissues are considered. It is noted that estimates for the elastic modulus of the individual trabecula of human cancellous bone vary from 1 to 20 GPa and it is suggested that this question needs further study.     

Isolation of genes encoding the Neurospora vacuolar ATPase. Analysis of vma-1 encoding the 67-kDa subunit reveals homology to other ATPases

The vacuolar membrane of Neurospora crassa contains a H+-translocating ATPase composed of at least three subunits with approximate molecular weights of 70,000, 60,000, and 15,000. Both genomic and cDNA clones encoding the largest subunit, which appears to contain the active site of the enzyme, have been isolated and sequenced. The gene for this subunit, designated vma-1, contains six small introns (60-131 base pairs) and encodes a hydrophilic protein of 607 amino acids, Mr 67,121. Within the sequence is a putative nucleotide-binding region, consistent with the proposal that this subunit contains the site of ATP hydrolysis. This 67-kDa polypeptide shows high homology (62% identical residues overall and 84% in the middle of the protein) to the analogous polypeptide of a higher plant vacuolar ATPase. The hypothesis that the vacuolar ATPase is related to F0F1 ATPases is strongly supported by the finding of considerable homology between the 67-kDa subunit of the Neurospora vacuolar ATPase and both the alpha and beta subunits of F0F1 ATPases.     

Short-term leaf elongation kinetics of maize in response to salinity are independent of the root

The essentiality of roots to the short-term responses of leaf elongation to salinity was tested by removing the roots of maize (Zea mays L.) from the shoots and comparing the initial short-term response of leaf elongation to that with intact plants. Eightday-old seedlings growing in solution culture were treated with 80 millimolar NaCl and their leaf elongation rate (LER) was monitored with a linear variable differential transformer connected to a computerized data aquisition system. Initially, LER of intact plants was sharply reduced by salinity, then rose rapidly to reach a new steady-state rate about 1.5 hours after salinization. The new steady-state rate of salinized intact plants was about 80% of the control rate. When the roots of nonsalinized plants were excised under the surface of the nutrient solution, excision did not disturb the steady-state LER. When these shoots were salinized, they responded in a manner nearly identical to that of intact plants, indicating that roots are not essential for the modulation of short-term LER of salt-stressed plants.