Purification and properties of microsomal UDP-glucuronosyltransferase from rat liver.

p-Nitrophenol conjugating activity associated with liver microsomal UDP-glucuronosyltransferase (EC 2.4.1.17) was purified 150- to 200-fold from cell-free homogenates. The purification scheme included solubilization with the nonionic detergent Lubrol WX, anion exchange chromatography at pH 6.0 and 7.5, and affinity chromatography with UDP-hexanolamine Sepharose 4B. The enzyme purified as a phospholipid-protein complex and was shown to consist of a single polypeptide chain of molecular weight 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis indicated approximately 531 mol of amino acids/59,000 g of enzyme and a molar ratio of nonpolar to polar residues of 1.08. During fractionation, the enzyme displayed instability with such steps as gel filtration, dialysis, or ultrafiltration of dilute samples; however, upon adsorption to ion exchange resins or storage in concentrated form, the enzyme was reasonably stable. The active lipoprotein complex showed both size and charge heterogeneity as judged by gel filtration and electrofocusing. Three forms of the enzyme resolved by isoelectric focusing had isoelectric points which averaged pH 6.68, 6.56, and 6.31. Polypeptide compositions of these electrophoretically distinct phospholipid protein complexes were indistinguishable on the basis of sodium dodecyl sulfate-polyacryl-amide gel electrophoresis, suggesting that the charge heterogeneity may be the result of differences in the phospholipid content of the lipoprotein complex.     

Cloning of multiple copies of immunoglobulin variable kappa genes in cosmid vectors

The possibility of cloning large segments of DNA in cosmid vectors offers distinct advantages, in particular for the study of multigene families. Large size fragments of mouse embryo DNA were successfully cloned in the cosmid pHC 79. Twelve recombinants hybridizing specifically to an immunoglobulin kappa chain variable region probe were identified. In 9 of these recombinants, the size of the insert ranges from 30 to 43 kilobases. Factors affecting the cloning efficiency of a complex mammalian genome in cosmids were studied. The stability of these recombinant cosmids and the preparation of recombinant cosmid DNA are also discussed.     

Antibodies directed toward human erythrocyte Ca2+-ATPase: effect on enzyme function and immunoreactivity of Ca2+-ATPases from other sources

Antibodies directed against purified human erythrocyte Ca²⁺-ATPase (purified according to a procedure modified from V. Niggli, J. T. Penniston, and E. Carafoli, 1979, J. Biol. Chem., 254, 9955–9958) were raised in rabbits. In competitive radioimmunoassay tests of immunological cross-reactivity, human erythrocyte Ca²⁺-ATPase shows a consistent pattern of immunological similarity to the Ca²⁺-ATPases derived from cell surface fractions of other species, such as rat and dog erythrocyte ghosts, rat corpus luteum plasma membranes, and rat brain synaptic plasma membranes. On the other hand, a purified Ca²⁺-ATPase preparation from rabbit skeletal muscle sarcoplasmic reticulum failed to show any immunological similarity to the human enzyme. The amount of Ca²⁺-ATPase protein in the erythrocyte ghosts was estimated to be about 0.6 μg/mg ghost protein, which was not too different from the calculated value of 1.2 ± 0.2 μg/mg ghost protein (mean ± SD, n = 6) based on the calmodulin binding studies of the erythrocyte ghosts. Anti-Ca²⁺-ATPase immunoglobulin G inhibited enzyme activity and calcium transport, showing that at least one subpopulation of antibodies can block the active site of the enzyme. The antibodies had no effect on the binding of calmodulin to erythrocyte membranes.     

A molecular model of RGD ligands. Antibody D gene segments that direct specificity for the integrin alpha IIb beta 3.

Following an ill-defined activation event, the Arg-Gly-Asp (RGD) recognition site of the platelet integrin, glycoprotein IIb-IIIa (alpha IIb beta 3), can bind to fluid-phase, RGD-containing protein ligands, such as fibrinogen, or to the murine monoclonal IgM, PAC-1, which contains the sequence Arg-Tyr-Asp (RYD) within the third complementarity-determining region of its heavy chain (H3). PAC-1 has thus become a widely exploited marker of platelet alpha IIb beta 3 activation. In this report, we compare PAC-1 with two murine IgG, OP-G2 (IgG1 kappa) and LJ-CP3 (IgG1 kappa), that also contain the sequence RYD in H3 but bind to alpha IIb beta 3 without prior activation. Each antibody can inhibit the binding of the other two to intact platelets or to purified IIb-IIIa, the binding of each antibody is completely inhibited by peptides containing RGD, and H3 of each antibody uses the germline D-gene DSP 2.10 (CTATAGGTACGAC) which includes the sequence RYD. Two other murine IgG, HP20 and PCG1-1, cloned and sequenced by other laboratories, also utilize the DSP 2.10 sequence, but neither antibody binds to alpha IIb beta 3. From a comparison of the H3 sequences of these antibodies, we have developed a molecular model of the H3 loop region which can explain these differences in specificity. This model predicts that both the ability to bind to alpha IIb beta 3 and the activation dependence of that binding are a function of the orientation and, therefore, accessibility of the RYD sequence. This model and refinements thereof can be exploited to study the molecular basis for specificity and affinity of RGD-containing ligands for integrins.     

Muscle triglyceride metabolism during exercise.

Skeletal muscle cell contains a considerable amount of triglycerides. The amount stored depends on the animal species as well as on muscle fiber composition. It is well documented that triglycerides in the fast-twitch red muscle and to a lesser extent in the slow-twitch muscle, but not those in the fast-twitch white muscle, are mobilized during prolonged exercise. Yet, little is known about the regulation of the metabolism of muscle triglycerides either at rest or during exercise. This is well reflected by the fact that an enzyme responsible for the hydrolysis of muscle triglycerides has not been identified. Mobilization of muscle triglycerides during exercise seems to be under both adrenergic and noradrenergic control. Accumulation of lactic acid and reduction in muscle pH are likely to be strong inhibitors of muscle triglyceride lipolysis. Reduction of carbohydrate availability accelerates mobilization of muscle triglycerides during exercise. The relationship between the plasma free fatty acids and muscle triglyceride metabolism seems to be complex. It has been proposed that most free fatty acids entering the muscle cell is esterified before being oxidized, but this is arguable for contracting skeletal muscles. It is suggested that most free fatty acids entering contracting high oxidative myocytes are transported directly to the mitochondria. A much lesser portion is likely esterified. It is proposed that triglycerides stored in the contracting muscle cell are mobilized when the delivery of the blood-borne-free fatty acids to the mitochondria is insufficient.     

Characterization of cDNAs encoding CuZn-superoxide dismutases in Scots pine

A Scots pine (Pinus sylvestris L.) cDNA library was screened with two heterologous cDNA probes (P31 and T10) encoding cytosolic and chloroplastic superoxide dismutases (SOD) from tomato. Several positive clones for cytosolic and chloroplastic superoxide dismutases were isolated, subcloned, mapped and sequenced. One of the cDNA clones (PS3) had a full-length open reading frame of 465 bp corresponding to 154 amino acid residues and showed approximately 85% homology with the amino acid sequences of angiosperm cytosolic SOD counterparts. Another cDNA clone (PST13) was incomplete, but encoded a putative protein with 93% homology to pea and tomato chloroplastic superoxide dismutase. The derived amino acid sequence from both cDNA clones matched the corresponding N-terminal amino acid sequence of the purified mature SOD isozymes. Northern blot hybridizations showed that, cytosolic and chloroplastic CuZn-SOD are expressed at different levels in Scots pine organs. Sequence data and Southern blot hybridization confirm that CuZn-SODs in Scots pine belong to a multigene family. The results are discussed in relation to earlier observations of CuZn-SODs in plants.     

Localization of DNA sequences to a region within Xp11.21 between incontinentia pigmenti (IP1) X-chromosomal translocation breakpoints.

Incontinentia pigmenti (IP) is an X-linked dominant disorder characterized by developmental anomalies of the tissues and organs derived from embryonic ectoderm and neuroectoderm. An IP locus, designated IP1, probably resides in Xp11.21, since five unrelated patients with nonfamilial IP have been identified who possess constitutional de novo reciprocal X;autosome translocations involving Xp11.21. We have used a series of somatic cell hybrids containing the rearranged chromosomes derived from three of the five IP1 patients, along with other hybrid cell lines, to map probes in the vicinity of the IP1 locus. Five anonymous DNA loci--DXS422, DXS14, DXS343, DXS429, and DXS370--have been mapped to a region within Xp11.21, between two IP1 X-chromosomal translocation breakpoints; the IP1 t(X;17) breakpoint is proximal (centromeric) to this region, and the IP1 t(X;13) and t(X;9) X-chromosomal breakpoints lie distal to it. While no IP1 translocation breakpoint has yet been identified by pulsed-field gel electrophoretic (PFGE) analysis, an overlap between three probes--p58-1, 7PSH3.5, and cpX210--has been detected, placing these probes within 125 kb. Four probes--p58-1, 7PSH3.5, cpX210, and 30CE2.8--have been helpful in constructing a 1,250-kb PFGE map of the region between the breakpoints; these results suggest that the IP1 X-chromosomal translocation breakpoints are separated by at least this distance. The combined somatic cell hybrid and PFGE analyses we report here favor the probe order DXS323-(IP1 t(X;13), IP1, t(X;9]-(DXS422, DXS14, DXS343, DXS429, DXS370)-(IP1 t(X;17), DXZ1). These sequences provide a starting point for identifying overlapping genomic sequences that span the IP1 translocation breakpoints; the availability of IP1 translocation breakpoints should now assist the cloning of this locus.     

Effects of neonatal septal lesions on reproductive behavior of male and female rats

The purpose of this study was to examine the effects of neonatally placed septal lesions (SL) in male, female, and androgenized female rats on reproductive behavior. Animals were castrated as adults and tested for both feminine and masculine sexual behavior. After treatment with estradiol benzoate (EB) alone (2 μg daily for 3 days), only the females with SL which had not been given testosterone propionate (TP) neonatally showed a facilitation of lordosis behavior. Following EB (2 μg for 3 days) plus 0.5 mg progesterone (P), both the lesioned and the sham-operated female groups showed an increase in the display of lordosis in either hormonal condition. All animals were given a pretest for masculine sexual behavior and tested on Days 4, 7, 11, and 15 of daily TP treatment (150 μg/day). There was no effect of the neonatally placed SL on masculine sexual behavior in female rats or in female rats androgenized with 30 μg TP. However, lesioned females treated neonatally with 1 mg TP showed a marginal enhancement of masculine sexual behavior. Male rats given SL neonatally showed a marked enhancement of masculine sexual behavior compared to that of controls. These results suggest that, depending on the neonatal hormone environment, SL selectively increase behavioral sensitivity to hormones. Although neonatally lesioned females show behavioral responses similar to females given SL as adults, male rats given SL neonatally are unique in that they show enhanced masculine sexual behavior whereas males lesioned as adults do not.     

Light Effects in Yeast: Evidence for Participation of Cytochromes in Photoinhibition of Growth and Transport in Saccharomyces cerevisiae Cultured at Low Temperatures

Visible light of moderate intensity inhibits growth, respiration, protein synthesis, and membrane transport in bakers' yeast and has a deleterious effect on membrane integrity. The results of this study indicate that these effects require the presence of cytochromes b and a/a(3). The light sensitivities of growth rate and [(14)C]histidine uptake in wild-type rho(+) Y185 and D225-5A strains of Saccharomyces cerevisiae were compared with those in a variety of mutants lacking cytochrome b or a/a(3) or both; a close correlation was found between the presence of these respiratory pigments and photosensitivity. Thus, strain TL5-3C, a nuclear petite lacking cytochromes b, a, and a(3), was resistant to light; strain GL5-6A, another nuclear petite having reduced amounts of cytochromes a and a(3), was partially resistant; strains MB127-20C and MB1-6C, nuclear petites lacking only cytochrome b, were also only partially resistant to light; whereas mutants containing all three cytochromes but having their respiratory chain either nonfunctional (strain ZK3-6B) or uncoupled (strain 18-27/t12) were fully sensitive to light. Finally, an equal-energy, broad-band action spectrum for the light inhibition of growth and transport indicated that blue light (408 nm) was most effective; these wavelengths correspond to the Soret region of the cytochrome absorption spectrum. The results suggest, therefore, that the yeast cytochromes b, a, and a(3) are the primary photoreceptors for the inhibitory effects of light and, perhaps, for other processes, such as the entrainment of biological rhythms in this species.