Increase of a form of UDP-glucuronyltransferase glucuronizing various phenolic xenobiotics and the corresponding translatable mRNA in 3-methylcholanthrene-treated rat liver

Induction of hepatic microsomal UDP-glucuronyltransferase activity toward various phenolic xenobiotics by 3-methylcholanthrene treatment of rats was observed, and the process of the induction was studied. We had previously purified a form of UDP-glucuronyltransferase (called GT-1) having a catalytic activity toward phenolic xenobiotics from liver microsomes of 3-methylcholanthrene-treated rats. The antibodies against GT-1 inhibited the enzyme activity toward those xenobiotics in liver microsomes, and bound to a single protein having a molecular weight of about 54,000 Da (same value as that of GT-1) among microsomal proteins on immunoblotting analysis. The amount of GT-1 protein in hepatic microsomes was found to be increased in close correspondence with the activity increase by 3-methylcholanthrene treatment, by immunoblotting analysis using an uninducible cytochrome P-450 reductase as a negative standard. It was shown by in vitro translation assays that the protein increase described above resulted from the enhancement of the level of translatable mRNA encoding for GT-1. Increases in the amount of the protein immunochemically corresponding to GT-1 in the microsomes from liver of phenobarbital-treated rats and from extrahepatic organs, such as kidney, small intestine, and lung, of phenobarbital- or 3-methylcholanthrene-treated rats were also observed.     

Isolation and characterization of major urinary amino acid O-glycosides and a dipeptide O-glycoside from a new lysosomal storage disorder (Kanzaki disease). Excessive excretion of serine- and threonine-linked glycan in the patient urine

Four major sialo compounds, termed GP-M1, GP-D1, GP-D2, and GP-D3 have been isolated from the urine of a novel glycoprotein storage disorder patient with angiokeratoma corporis diffusum which was discovered by Kanzaki et al. (Kanzaki, T., Yokota, M., Mizuno, N., Matsumoto, Y., and Hirabayashi, Y. (1989) Lancet April 22, 875-877). Based on the results of fast atom bombardment mass spectrometry, methylation analysis, and proton nuclear magnetic resonance spectroscopy, their chemical structures were concluded to be: (formula; see text) The yields of GP-M1, GP-D1, GP-D2, and GP-D3 were approximately 15, 6, 50, and 5 mg/liter of urine, respectively. The most major compound GP-D2, was further purified into single molecular species, threonine and serine type, by reversed phase high performance liquid chromatography. NMR analysis of the two purified compounds with single molecular species showed that the chemical shifts of anomeric protons of GalNAc were significantly different between threonine- and serine-linked GalNAc. Neither mannose-containing glycopeptides nor glycosphingolipids were excreted in the patient urine. From these results, this disease is thought to be caused by the deficiency of a lysosomal enzyme(s) acting on O-linked glycan chains.     

A naturally occurring mutation of insulin receptor alanine 1134 impairs tyrosine kinase function and is associated with dominantly inherited insulin resistance

We have identified a previously undescribed genetic variant of the insulin receptor (Ala1134----Thr1134) in a family with the Type A syndrome of insulin resistance. Using the polymerase chain reaction to amplify insulin receptor cDNA and genomic DNA (exon 19), this mutation was detected in 1/2 alleles in the proband, her two affected sisters, and her affected father. Two normal alleles were present in the unaffected mother. No additional structural changes were encoded by the remainder of the proband's receptor cDNA. The Ala1134 mutant receptor was expressed in Chinese hamster ovary cells. The expressed mutant receptors were processed normally and displayed normal affinity of insulin binding but were markedly deficient in insulin-stimulated autophosphorylation. The mutant receptor was unable to catalyze the phosphorylation of the endogenous substrate, pp185, and insulin-stimulated kinase activity toward an exogenous substrate in vitro also was markedly impaired. Ala1134 is a highly conserved residue located in a consensus sequence found in most tyrosine kinases. It is likely that this previously uncharacterized residue and/or the immediate region surrounding it are important for normal kinase function in other members of this receptor family. This study also demonstrates that severe insulin resistance with dominant inheritance may be caused by a missense mutation in one allele of the insulin receptor gene.     

Application of immobilized lipase to regio-specific interesterification of triglyceride in organic solvent

Summary Lipase from Rhizopus delemar was immobilized by entrapment with photo-crosslinkable resin prepolymers or urethane prepolymers or by binding to various types of porous silica beads. The immobilized lipase preparations thus obtained were examined for their activity in converting olive oil to an interesterified fat (cacao butter-like fat), whose oleic acid moieties at 1- and 3-positions were replaced with stearic acid moieties, in the reaction solvent n-hexane. Although all of the immobilized preparations exhibited some activity, lipase adsorbed on Celite and then entrapped with a hydrophobic photo-crosslinkable resin prepolymer showed the highest activity, about 75% of that of lipase simply adsorbed onto Celite. Entrapment markedly enhanced the operational stability of lipase.Dedicated to Professor H. Holzer, Freiburg University, on his 60th birthday (June 13, 1981)     

Post-embryonic development of circadian rhythm in the cricket,Gryllus bimaculatus: A rhythm reversal

Summary Locomotor activity of the male cricketGryllus bimaculatus DeGeer was recorded from the 7th or last (8th) instar nymph. The nymph showed a diurnal rhythm (nymphal rhythm = NR), while the adult, on the contrary, was nocturnal (adult rhythm = AR) (Fig. 1). This rhythm reversal occurred suddenly 3 to 5 days after the imaginal molt, almost simultaneously with the first spermatophore formation and the start of stridulation (calling song) (Fig. 2). In addition to the antiphase relationship, both rhythms also differed in the freerunning period (tau) and wave form. Tau_scdd was significantly longer in NR (24.33 h) than in AR (23.91 h) (Fig. 3). AR was characterized by a sharp activity peak in each cycle, which NR, however, lacked (Fig. 1, 3, 6). On the basis of these differences, two possibilities are discussed; one is that NR and AR are separate oscillations and the other is that both are coupled to different phase points of one oscillation.Abbreviations LD light dark - DD constant darkness - LL constant light - NR nymphal rhythm - AR adult rhythm     

Preparation of Uridine Diphosphate-N-Acetylgalactosamine from Uridine Diphosphate-N-Acetylglucosamine by Using Microbial Enzymes

A method was developed for the large scale preparation of uridine diphosphate-N-acetylgalactosamine (UDP-GalNAc) from uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) by means of microbial enzymes. With Bacillus subtilis cell-free extract as a source of UDP-GlcNAc 4-epimerase, about 35% of the UDP-GlcNAc added was converted to UDP-GalNAc. After the residual UDP-GlcNAc was degraded to uridine triphosphate and N-acetylglucosamine-1-phosphate with a protamine-treated extract of bakers' yeast as a source of UDP-GlcNAc pyrophosphorylase, UDP-GalNAc was separated by anion-exchange column chromatography. The nucleotide was recovered by adsorption on charcoal and elution with ammoniacal ethanol. The final yield was about 100 μmol.     

Antioxidative effect of α-tocopherol incorporation into lecithin liposomes on ascorbic acid-Fe2+-induced lipid peroxidation

The antioxidative effect of α-tocopherol incorporated into lecithin liposomes was studied. Lipid peroxidation of liposome membranes, assayed as malondialdehyde production, was catalyzed by ascorbic acid and Fe²⁺. The peroxidation reaction, which did not involve the formation of singlet oxygen, superoxide, hydrogen peroxide, or a hydroxyl radical, was inhibited by α-tocopherol and a model compound of α-tocopherol, 2,2,5,7,8-pentamethyl-6-hydroxy-chroman (TMC), but not by phytol, α-tocopherylquinone, or α-tocopheryl acetate. One mole of α-tocopherol completely prevented peroxidation of about 100 moles of polyunsaturated fatty acid. Decrease in membrane fluidity by lipid peroxidation, estimated as increase of fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) embedded in the membrane, was also inhibited by α-tocopherol and TMC, reflecting their antioxidant functions. Cholesterol did not act as an antioxidant, even when incorporated in large amount into the liposome membranes, but it increased the antioxidative efficiency of α-tocopherol. When a mixture of liposomes with and without α-tocopherol was incubated with Fe²⁺ and ascorbic acid, α-tocopherol did not protect the liposomes not containing α-tocopherol from peroxidation. However, preincubation of the mixture, or addition of Triton X-100 allowed the α-tocopherol to prevent peroxidation of the liposomes not containing α-tocopherol. In contrast, in similar experiments, liposomes containing TMC prevented peroxidation of those without TMC without preincubation. Tocopherol in an amount so small as to exhibit only a slight antioxidative effect was oxidized when incorporated in egg lecithin liposomes, but it mostly remained unoxidized when incorporated in dipalmitoyllecithin liposomes, indicating that oxygen activated by ascorbic acid-Fe²⁺ does not oxidize α-tocopherol directly. Thus, decomposition of α-tocopherol may be caused by its interaction with peroxy and/or alkoxyl radicals generated in the process of lipid peroxidation catalyzed by Fe²⁺ and ascorbic acid.     

Electron transfer between horse heart and Candida krusei cytochromes c in the free and bound states

Electron transfer between horse heart and Candida krusei cytochromes c in the free and phosvitin-bound states was examined by difference spectrum and stopped-flow methods. The difference spectra in the wavelength range of 540–560 nm demonstrated that electrons are exchangeable between the cytochromes c of the two species. The equilibrium constants of the electron transfer reaction for the free and phosvitin-bound forms, estimated from these difference spectra, were close to unity at 20°C in 20 mM Tris-HCl buffer (pH 7.4). The electron transfer rate for free cytochrome c was (2–3) · 10⁴ M^?1 · s^?1 under the same conditions. The transfer rate for the bound form increased with increase in the binding ratio at ratios below half the maximum, and was almost constant at higher ratios up to the maximum. The maximum electron exchange rate was about 2 · 10⁶ M^?1 · s^?1, which is 60–70 times that for the free form at a given concentration of cytochrome c. The activation energy of the reaction for the bound cytochrome c was equal to that for the free form, being about 10 kcal/mol. The dependence of the exchange rate on temperature, cytochrome c concentration and solvent viscosity suggests that enhancement of the electron transfer rate between cytochromes c on binding to phosvitin is due to increase in the collision frequency between cytochromes c concentrated on the phosvitin molecule.     

Solubilization and partial purification of protein kinase systems from brain membranes that phosphorylate calspectin: A spectrin-like calmodulin-binding protein (fodrin)

In brain tissue a spectrin-like calmodulin-binding protein calspectin, or fodrin, is concentrated in a synaptosome fraction, where most of the calspectin is associated with the synaptic membranes. This endogenous calspectin was phosphorylated by protein kinase system(s) associated with the membranes. Here, we report the solubilization and partial purification of the membrane-associated calspectin kinase activity. The activity was resolved on a gel filtration column into two fractions, peaks I and II having estimated M_r of 800 000 and 88 000. The activity of peak I was dependent on the presence of both Ca²⁺ and calmodulin. Peak II revealed a basal activity in the absence of Ca²⁺ and calmodulin, which was stimulated 2-fold by addition of Ca²⁺. Calmodulin had no effect on the peak II activity.     

Release of heat-labile enterotoxin subunits by Escherichia coli.

Most of the heat-labile enterotoxin (LT) synthesized by Escherichia coli is cell associated; however, a small portion of LT (approximately 10%) is released by bacterial cells into the culture supernatant. The LT subunit B (LT-B) produced by a cloned LT-B gene (tox B) was released in amounts equal to the parent LT release. In contrast, no release of LT subunit A (LT-A) or its smaller derivatives was observed in strains containing cloned toxA genes. The data suggest that LT-B is necessary for the release of LT-A across the bacterial membrane.