A ubiquitin mutant with specific defects in DNA repair and multiubiquitination.

The degradation of many proteins involves the sequential ligation of ubiquitin molecules to the substrate to form a multiubiquitin chain linked through Lys-48 of ubiquitin. To test for the existence of alternate forms of multiubiquitin chains, we examined the effects of individually substituting each of six other Lys residues in ubiquitin with Arg. Substitution of Lys-63 resulted in the disappearance of a family of abundant multiubiquitin-protein conjugates. The UbK63R mutants were not generally impaired in ubiquitination, because they grew at a wild-type rate, were fully proficient in the turnover of a variety of short-lived proteins, and exhibited normal levels of many ubiquitin-protein conjugates. The UbK63R mutation also conferred sensitivity to the DNA-damaging agents methyl methanesulfonate and UV as well as a deficiency in DNA damage-induced mutagenesis. Induced mutagenesis is mediated by a repair pathway that requires Rad6 (Ubc2), a ubiquitin-conjugating enzyme. Thus, the UbK63R mutant appears to be deficient in the Rad6 pathway of DNA repair. However, the UbK63R mutation behaves as a partial suppressor of a rad6 deletion mutation, indicating that an effect of UbK63R on repair can be manifest in the absence of the Rad6 gene product. The UbK63R mutation may therefore define a new role of ubiquitin in DNA repair. The results of this study suggest that Lys-63 is used as a linkage site in the formation of novel multiubiquitin chain structures that play an important role in DNA repair.     

Two steps in the transition between the native and acid states of bovine α-lactalbumin detected by circular polarization of luminescence: Evidence for a premolten globule state?

A few studies indirectly support the existence of an intermediate in the transition of Ca(2+)-saturated bovine alpha-lactalbumin (alpha-LA) from the native (N) to the acidic (A) state, known as the molten globule state. However, direct experimental evidence for the appearance of this intermediate has not been obtained. The signal of circular polarization of luminescence (CPL) is sensitive to fine conformational transitions because of its susceptibility to changes in the environmental asymmetry of fluorescent chromophores in their excited electronic states. In the present study, CPL measurements were applied using the intrinsic tryptophan fluorescence of alpha-LA as well as the fluorescence of 8-anilino-1-naphthalenesulfonic acid (ANS) bound to alpha-LA. CPL of tryptophan and ANS was measured in the pH range of 2.5-6 in order to find direct experimental evidence for the proposed intermediate. CPL (characterized by the emission anisotropy factor, g(em)) depends on the asymmetry of the protein molecular structure in the environment of the tryptophan and the ANS chromophores in the excited electronic state. The pH dependence of both the gab, absorption anisotropy factor determined by CD, and the ANS steady state fluorescence, showed a single transition at pH 3-3.7 as already reported elsewhere. This transition was interpreted as being a result of a change of the alpha-LA tertiary structure, which resulted in a loss of asymmetry of the environment of both the tryptophan residues and the ANS hydrophobic binding sites. The pH dependence of the tryptophan and ANS g(em) showed an additional conformational transition at pH 4-5, which coincided with the pKa of Ca2+ dissociation (pKa 5), as predicted by Permyakov et al. (1981, Biochem Biophys Res Commun 100:191-197). The titration curve showed that there is a pH range between 3.7 and 4.1 in which alpha-LA exists in an intermediate state between the N- and A-state. We suggest that the intermediate is the premolten globule state characterized by a reduced Ca2+ binding to the alpha-LA, native-like tertiary structure, and reduced asymmetric fluctuation of the tertiary structure on the nanosecond time scale. This intermediate resembles the "critical activated state" theoretically deduced by Kuwajima et al. (1989, J Mol Biol 206:547-561). The present study demonstrates the power of CPL measurements for the investigation of folding/unfolding transitions in proteins.     

Microcomputer interface for computer-controlled enzyme kinetic studies with the monolayer technique

Periodic acid oxidation in methanol followed by incubation with 1, 1-dimethylhydrazine results in release of diacylglycerols from 1,2-diacyl-3-glycosyl-sn-glycerols. During hydrazinolysis of oxidized monogalactosyl diacylglycerol, an intermediate hydrazone derivative was observed which was isolated and identified. The diacylglycerols recovered are 1,2-diacyl isomers containing the same fatty acid mixtures as the intact glycolipids. The yields of diacylglycerols released from plant monogalactosyl-, digalactosyl-, and sulfoquinovosyl diacylglycerols were in the range of 30-50%. The method may be used for analysis of molecular species and for preparative purposes.     

Apolipoprotein A-IGiessen (Pro143----Arg). A mutant that is defective in activating lecithin:cholesterol acyltransferase

Apolipoprotein A-IGiessen is a variant form of apo A-I that is displaced from the corresponding normal A-I isoforms on isoelectric focusing gels by a single charge unit towards the cathode [Utermann et al. (1982) J. Biol. Chem. 257, 501-507]. Three subjects heterozygous for the variant were detected in one family. The percentage of the total A-I in plasma represented by the A-IGiessen in these subjects ranged over 25-30%. The variant and normal major A-I isoforms from the proband (Y.J.) were purified by preparative isoelectric focusing and cleaved with CNBr. Analytical focusing of CNBr fragments demonstrated a charge difference between CB3Giessen and normal CB3. Sequence analysis of CB3Giessen revealed that a proline existing in normal A-I was replaced by an arginine in the variant A-I at residue 143. The ability of the mutant A-I to activate purified lecithin:cholesterol acyltransferase was determined in vitro. The cofactor activity of [Arg143]apolipoprotein A-I was about 60-70% of that demonstrated by control A-I. Residue 143 is in a putative beta-turn between two of the repeating amphiphilic helices in apolipoprotein A-I and may be a critical determinant of the protein's structure and function.     

Refined mapping of the gene for glutathione reductase on human chromosome 8

Summary Activity of the enzyme glutathione reductase (EC 1.6.4.2) in erythrocytes and fibroblasts of a patient with karyotype 46, XY, del(8) (pterp212:) was found to be in the normal range. With results from other laboratories, this allowed a more precise mapping of the gene for this enzyme in the region 8p2100–8p212.     

Composition of soluble cuticular lipids and water permeability of cuticular membranes from Citrus leaves

Water permeability and composition of soluble cuticular lipids of isolated cuticular membranes from leaves of Citrus aurantium L. were investigated for 3 successive years. The average water permeability coefficient determined using 169 cuticular membranes was 1.09·10^–7 cm s^–1 with a standard deviation of 0.78·10^–7 cm s^–1. There were no significant differences in water permeability between years. Cuticular membranes are characterized by a great variability in water permeability both within and between years. Both water permeability of individual membranes and variability between membranes are shown to be determined by soluble cuticular lipids contained within the cuticular membranes. The soluble cuticular lipids of Citrus leaves are composed of fatty acids, primary alcohols, esters, and hydrocarbons. They occur in amounts of 9.84 g cm^–2, which represents approx. 3% of the total mass of isolated cuticular membranes. The specific weight of cuticular membranes (365.4 g cm^–1) and total amount of soluble cuticular lipids did not vary significantly between years. Significant differences were observed for the amounts and composition of the constituent classes of lipids. Six homologues comprise 86% of the fatty acids (C₁₆; C₁₈; C₁₉; C₂₁; C₂₄; C₂₆), 83% of the primary alcohols (C₂₄; C₂₆; C₂₈; C₃₀; C₃₂; C₃₄) and 88% of the esters (C₃₆; C₃₈; C₄₀; C₄₁; C₄₂; C₄₄). Eleven major homologues amount only to 62% of the total hydrocarbons (C₁₆; C₁₇; C₁₈; C₂₀; C₂₆; C₂₇; C₂₉; C₃₀; C₃₁; C₃₂; C₃₃). Variability in the composition of soluble cuticular lipids between years was much smaller than variability of water permeability and, therefore, no relation between composition of soluble cuticular lipids and water permeability could be found. It is suggested that this may be due to the fact that the lipid composition observed represents the averages of 20 to 30 membranes analyzed so that differences between individual membranes may have been leveled out.Abbreviations CM cuticular membranes - MX polymer matrix - P_d permeability coefficient for diffusion of water - SCL soluble cuticular lipids - MES morpholinoethane sulphonic acid     

Leukemogenic activity of thymotropic, ecotropic, and xenotropic radiation leukemia virus isolates.

Thymotropic, ecotropic, and xenotropic oncoviruses were isolated from the C57BL/6 mouse radiation leukemia system and were propagated in culture. The purified viruses were inoculated singly and in various combinations into groups of mice, and leukemia incidence was determined. Only the thymotropic virus was leukemogenic in vivo.     

Ubiquitin conjugation by the yeast RAD6 and CDC34 gene products. Comparison to their putative rabbit homologs, E2(20K) AND E2(32K).

The recombinant yeast RAD6 and CDC34 gene products were expressed in Escherichia coli extracts and purified to apparent homogeneity. The physical and catalytic properties of RAD6 and CDC34 were similar but distinct from their putative rabbit reticulocyte homologs, E2(20k) and E2(32k), respectively. Like their reticulocyte counterparts, RAD6 and CDC34 are bifunctional enzymes competent in both ubiquitin:protein ligase (E3)-independent and E3-dependent conjugation reactions. RAD6 and E2(20k) exhibit marked specificity for the conjugation of core histones and catalyze the processive ligation of up to three ubiquitin moieties directly to such model substrates. RAD6 differed from its putative E2(20k) homolog in exhibiting simple saturation behavior in the kinetics of histone conjugation and in being unable to distinguish kinetically between core histones H2A and H2B, yielding identical values of kcat (1.9 min-1) and Km (20 microM). A slow rate of multiubiquitination involving formation of extended ubiquitin homopolymers on the histones was also observed with RAD6 and E2(20k). Comparison of conjugate patterns among native, reductively methylated, and K48R ubiquitin variants demonstrated that the linkage between ubiquitin moieties formed by E2(20k) and RAD6 was not through Lys-48 of ubiquitin, the site previously demonstrated as a strong signal for degradation of the target protein. In contrast, CDC34 differs from its putative homolog, E2(32k), in showing a specificity for conjugation to bovine serum albumin rather than to core histones. Both CDC34 and E2(32k) exhibit a marked kinetic selectivity for processive multiubiquitination via Lys-48 of ubiquitin. Calculations based on a model ubiquitin conjugation reaction indicated that E2(32k) and CDC34 preferentially catalyzed multiubiquitination over ligation of the polypeptide directly to target proteins. Formation of such multiubiquitin homopolymers by E2(32k) and CDC34 suggests these enzymes may commit their respective target proteins to degradation via an E3-independent pathway.