Mechanism of ubiquitin carboxyl-terminal hydrolase. Borohydride and hydroxylamine inactivate in the presence of ubiquitin

Ubiquitin (Ub) carboxyl-terminal hydrolase (E) catalyzes the hydrolysis, at the Ub-carboxyl terminus, of a wide variety of C-terminal Ub derivatives. We show that the enzyme is inactivated by millimolar concentrations of either sodium borohydride or hydroxylamine, but only if Ub is present. We have interpreted these results on the assumption that the hydrolase mechanism is one of nucleophilic catalysis with an acyl-Ub-E intermediate. The borohydride-inactivated enzyme has the following properties. It is a stoichiometric complex of E and Ub containing tritium from sodium boro[3H]hydride. This complex is stable at neutral pH in 5 M urea and can be isolated on the basis of size on a sieving column, but a labeled product the size of Ub is released under more strongly denaturing conditions. The "Ub" released in acid is Ub-carboxyl-terminal aldehyde, based on the observations that: it contains the tritium present in the reduced complex and it is able to form the inactive enzyme from a stoichiometric amount of fresh enzyme, and inactivation is accompanied by E-Ub adduct formation; it has chemical properties expected of an aldehyde: after a second reduction of the Ub released with boro[3H]hydride and complete acid hydrolysis, tritium counts are found in ethanolamine (the carboxyl-terminal residue of Ub is glycine). These results suggest that enzyme and Ub combine in an equilibrium reaction to form an ester or thiol ester adduct (at the Ub-carboxyl terminus), and that this adduct is trapped by borohydride to give a very stable inactive E-Ub (thio) hemiacetal which is unable to undergo a second reduction step and which can release Ub-aldehyde in mild acid. Inactivation in the presence of hydroxylamine of hydrolase occurs once during hydrolysis of 1200 molecules of Ub-hydroxamate by the enzyme. The hydrolysis/inactivation ratio is constant over the range of 10-50 mM hydroxylamine showing that forms of E-Ub with which hydroxylamine and water react are different and not in rapid equilibrium. The inactive enzyme may be an acylhydroxamate formed from an E-Ub mixed anhydride generated from the E-Ub (thiol) ester inferred from the borohydride study. A direct radioactive assay for the hydrolase has been developed using the Ub-C-terminal amide of [3H]butanol-4-amine as substrate.     

N protein is the predominant antigen recognized by vesicular stomatitis virus-specific cytotoxic T cells.

The specificity of anti-vesicular stomatitis virus (VSV)-specific cytotoxic T cells was explored with cell lines expressing VSV genes introduced by electroporation. Low levels of nucleocapsid (N) protein were detected on the surface of VSV-infected cells, but N protein could not be detected on the plasma membrane of transfected EL4 cells. Intracellular N protein was detectable by enzyme-linked immunosorbent assay or immunoprecipitation in some of the transfected cell lines but not in others, unless the transfected genes were induced by sodium butyrate. However, all of the stably transfected EL4 cell lines expressing the VSV-Indiana N protein were efficiently lysed by serotype-specific and cross-reactive anti-VSV cytotoxic T cells (CTLs). Primary cross-reactive anti-VSV CTLs appeared to be specific solely for N protein, based on cold-target competition assays using infected and transfected target cells. Cell lines expressing 100- to 1,000-fold less N protein than did VSV-infected cells were efficiently lysed by both primary and secondary anti-VSV CTLs. Cell lines expressing 100-fold less G protein than did VSV-infected cells were not lysed by either population of effectors. Significantly, cold-target competition studies with secondary CTLs demonstrated that N protein-expressing cell lines were more efficient competitors than were VSV-infected cells even though the latter expressed 100- to 1,000-fold more N protein. This was not an artifact of viral infection since infection of the transfected cell lines did not affect their ability to compete. The possibility that cell lines constitutively expressing internal virus proteins present antigen more effectively than infected cells do is discussed.     

Female rats are protected against the fructose induced mortality of copper deficiency

Experiments were conducted in copper deficient male and female rats fed diets containing fructose or starch in order to determine whether the same type of interaction between copper status and dietary carbohydrate found in male rats also occurs in the female rat. Mortality occurred only in the male rats fed the fructose diet deficient in copper with 40% of the animals dying during the 8 week study. Only anemia, hypercholesterolemia, increased BUN, heart hypertrophy and reduced body weight were observed in these animals which could be related to their mortality. Despite the increased mortality, plasma ceruloplasmin, erythrocyte SOD and hepatic copper concentrations were reduced to a similar extent in all rats regardless of the sex of the animals or of the type of dietary carbohydrate fed. The results of the present study indicate that although direct measurements of copper status of female rats fed fructose diet deficient in copper are similar to their male counterpart, they are apparently protected from the lethal consequences of the deficiency.     

Development of copper deficiency in rats fed fructose or starch: weekly measurements of copper indices in blood

Copper deficiency was induced in weanling rats fed diets whose sole source of carbohydrates was starch or fructose for 7 weeks. Conventional parameters of copper status, plasma copper concentrations, ceruloplasmin activity, and erythrocyte superoxide dismutase (SOD) activity were longitudinally monitored weekly to follow the development of the deficiency and to correlate these indices with the degree of severity of the deficiency. Although 30% of the rats fed a copper-deficient fructose diet died and no deaths occurred in rats fed the copper-deficient starch diet, plasma copper, ceruloplasmin, and SOD activities were reduced to a similar extent in all rats fed copper-deficient diets regardless of the type of dietary carbohydrate. Thus, none of the indices used accurately reflected the greater degree of deficiency or mortality in rats fed the fructose diet deficient in copper. The results of the present study underscore the need for more sensitive tests or alternative parameters to assess copper status in living animals.     

G1m(1) and G1m(2) allotypes in Albanian towns of Calabria

The G1m(1) and G1m(2) allotype distribution was analyzed in a population sample from 11 Albanian towns of Calabria. The unusually high frequency of the G1m(1) marker already observed in Calabria as well as the presence of the Gm(2) phenotype were shown. The Calabrian and Albanian populations were similar, but significantly different from other Italian populations.     

Uptake of radiolabeled copper from portal blood containing fructose or glucose

The present investigation was designed to study the uptake of⁶⁷Cu when administered directly, into the portal vein, along with either functose or glucose, by the liver and extrahepatic tissues. Following weaning, male Sprague-Dawley rats were fed for 3 wk either commercial laboratory ration (chow) or semipurified diets deficient in Cu (0.6 ppm) or supplemented with Cu (6.0 ppm) and containing 62% carbohydrate as either fructuse or cornstarch. After an overnight fast, a single dose of rat plasma (0.1 mL) containing fructose or glucose extrinsically labeled with⁶⁷Cu was injected directly into their portal vein. Although not always statistically significant, rats fed chow retained more radioactivity in the liver and several extrahepatic tissues when⁶⁷Cu was administered with fructose than with glucose. Regardless of Cu status, rats fed diets containing fructose retained more radioactivity in extrahepatic tissues than rats fed starch. There was an increased uptake of⁶⁷Cu by the liver, blood, muscle, and fat pad when fructose as compared to glucose was injected in combination with the isotope. These data strongly suggest that Cu requirements or utilization are greater when fructose is the main dietary carbohydrate. The results may also in part explain the reason for the increased severity of Cu deficiency in rats fed fructose.     

Linkage of cystic fibrosis locus and polymorphic DNA markers in 14 families

Linkage relationships between the cystic fibrosis (CF) locus and three polymorphic DNA markers were examined in 14 families, five of which were of Hispanic origin. Tight linkage was found between the CF locus and MET (maximum lod score = 7.16 at theta = .001), and between CF and pJ3.11 (maximum lod score = 3.87 at theta = .001). We observed two recombinations between CF and collagen, yielding a maximum lod score of 0.359 at theta = .125, and one recombination in the cluster CF-MET-pJ3.11. Analysis by the seriation method indicates the order COL-pJ3.11-CF-MET.     

The enzymic synthesis of ribose-1,5-bisphosphate: studies of its role in metabolism

Ribose-1,5-bisphosphate is synthesized in a reaction that uses ribose-1(or 5)-P as the phosphoryl acceptor and the acyl-P of 3-phosphoglyceryl phosphate as the donor. Glucose-1,6-bisphosphate is synthesized in a similar reaction. The relative activity with the two substrates remains unchanged over almost 300-fold purification of the enzyme, indicating that glucose-1,6-bisphosphate synthase catalyzes both reactions. The relative V/Km values for alternative phosphoryl acceptors are ribose-1-P (1); glucose-1-P (0.30); mannose-1-P and ribose-5-P (0.11); glucose-6-P (0.10); 2-deoxyglucose-6-P (0.03); and 2-deoxyribose-5-P (0.02). Fructose-1- and 6-phosphates are not substrates. The synthesis of both ribose-1,5-bisphosphate and glucose-1,6-bisphosphate is inhibited by physiologically significant levels of fructose-1,6-bisphosphate, glycerate-2,3-bisphosphate, glycerate-3-phosphate, citrate, and inorganic phosphate. Ribose-1,5-bisphosphate is a strong activator of brain phosphofructokinase.     

Plasma cholesterol concentration and death from coronary heart disease: 10 year results of the Whitehall study.

Ten year mortality from coronary heart disease in 17,718 middle aged men was related to their initial plasma cholesterol concentrations. The relative risk of death from coronary heart disease declined with age, but the absolute excess risk did not. The risk gradient was continuous over the whole range of cholesterol concentrations, the lowest mortality being among men with concentrations below the lowest decile. It seems that, as with blood pressure, the average cholesterol concentration in the blood pressure, the average cholesterol concentration in the population is too high: lowest concentrations are prognostically the best. A quarter of all deaths from coronary heart disease related to cholesterol occurred among men with concentrations above the top decile, but 55% occurred among men with concentrations in the middle three fifths of the distribution; this figure of 55% could be reduced only by a policy aimed at lowering concentrations in the whole population.     

Blockade of NK cell lysis is a property of monoclonal antibodies that bind to distinct regions of T-200

The previously described NK inhibitory monoclonal antibody 13.1 is shown to immunoprecipitate a series of high m.w. glycoproteins homologous with the murine T-200/Ly-5 molecules. Not all antibodies to the human T-200 molecule, however, have an inhibitory effect on NK cell function. A comparison is made between two noninhibitory anti-T-200 antibodies, 13.5 and 13.6, and two inhibitory anti-T-200 antibodies, 13.1 and 13.3. All antibodies are of the IgG1 subclass. Sequential immunoprecipitation experiments show that these antibodies react with the same set of molecules. The differences in NK-blocking activity could not be explained by the amount of antibody bound per cell in NK-enriched populations, nor by the avidity with which they bound. It is shown by competitive radiobinding assays that the 13.1 and 13.3 antibodies define a region, termed region A, distinct from that defined by the nonblocking antibodies 13.5 and 13.6, termed region B. Region B is shown to reside between the membrane and region A. These findings show that the inhibition of NK lysis by anti-T-200 antibodies is a function of the site on that molecule to which these antibodies bind. This may also explain the ability of antibodies to the A region of T-200 to block selectively the lysis of myeloid and erythroid tumor targets, with no effect on the lysis of T lymphoma targets.