Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family

Background

Protein translocation across the membrane of the Endoplasmic Reticulum (ER) is the first step in the biogenesis of secretory and membrane proteins. Proteins enter the ER by the Sec61 translocon, a proteinaceous channel composed of three subunits, α, β and γ. While it is known that Sec61α forms the actual channel, the function of the other two subunits remains to be characterized.

Results

In the present study we have investigated the function of Sec61β in Drosophila melanogaster. We describe its role in the plasma membrane traffic of Gurken, the ligand for the Epidermal Growth Factor (EGF) receptor in the oocyte. Germline clones of the mutant allele of Sec61β show normal translocation of Gurken into the ER and transport to the Golgi complex, but further traffic to the plasma membrane is impeded. The defect in plasma membrane traffic due to absence of Sec61β is specific for Gurken and is not due to a general trafficking defect.

Conclusion

Based on our study we conclude that Sec61β, which is part of the ER protein translocation channel affects a post-ER step during Gurken trafficking to the plasma membrane. We propose an additional role of Sec61β beyond protein translocation into the ER.     

Kinetics of multiple transport systems for amino acids in the brain

Two models of multiple transport systems (one comprising diffusion plus a Michaelis-Menten component, and the other comprising two Michaelis-Menten components) were tested for their fit to a series of experimental data. A statistical method (linearization technique) was used to get estimates for the appropriate parameters of each model. In most cases the data could be fitted to either model. Techniques for discriminating between the two models were sought and are discussed.     

Essential sulfhydryl groups of rat liver monoamine oxidase.

The inhibition by some thiol reagents of partly purified mitochondrial monoamine oxidase (MAO) (EC 1.4.3.4) from rat liver was studied, and the molar content of sulfhydryl groups in the enzyme determined. Sodium nitroprusside and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) inhibited the enzyme, apparently reversibly, while sodium arsenite was not inhibitory. Concentrations of the respective inhibitors causing 50% inhibition after 15 min of preincubation with the enzyme at pH 7.0 and 37 degrees C are 5.80 times 10(-4) M and 4.35 times 10(-5) M. The thiol compounds cysteine, dithiothreitol, and 2-mercaptoethanol did not inhibit MAO. The average number of sulfhydryl groups per mole of enzyme, determined by reaction with DTNB, increased from 3.6 +/- 0.2 freely reacting sulfhydryl groups (n = 4) to 18.4 to total sulfhydryl groups (n = 2) on denaturation with 8 M urea.     

Tryptophan catabolism by tryptophan pyrrolase in rat liver. The effect of tryptophan loads and changes in tryptophan pyrrolase activity.

We investigated how changes in tryptophan pyrrolase activity and tryptophan loads affect the breakdown of tryptophan was estimated by injecting rats with [ring-2-14-C]tryptophan and measuring respiratory 14-CO2. We concluded, contrary to previous reports, that induction of tryptophan pyrrolase definitely will increase the rate of tryptophan breakdown. Tryptophan loads also increase tryptophan breakdown even in circumstances where there is no increase in tryptophan pyrrolase activity, presumably by increasing the saturation of the enzyme. After a tryptophan load (50 mg per kg) the increase in liver tryptophan concentration lasts only 30 min. The rapid return of liver tryptophan to normal may be due partly to the high turnover rate of liver tryptophan. We estimate that tryptophan pyrrolase degrades tryptophan in vivo at a rate that is equivalent to the whole liver tryptophan concentration in 7.5 min or less.     

Increased activity of spermidine N1-acetyltransferase in the adrenal cortex of rats following administration of exogenous spermidine, carbamylcholine, 2-deoxyglucose and dopamine agonists

The activity of N1-acetyltransferase was increased in the dissected adrenal cortex of the rat following a single administration of spermidine. The activity was maximal 6-8 h after the onset of treatment. The increase in enzyme activity was abolished when the rats were given cycloheximide 2 h after spermidine; this suggests that increased activity results from an augmentation in the synthesis of the enzyme. Adrenocortical spermidine N1-acetyltransferase was also induced by carbamylcholine, 2-deoxyglucose, apomorphine and piribedil, drugs that are known to cause induction of ornithine decarboxylase in that organ. Hypophysectomized rats showed reduced activity of spermidine N1-acetyltransferase when compared to sham-operated controls, and carbamylcholine no longer elicited an increase in enzyme activity in such animals. Adrenocortical spermidine N1-acetyltransferase activity of hypophysectomized rats is induced by corticotropin (ACTH). These results suggest a hormonal control over the activity of the enzyme in the adrenal cortex with ACTH acting as a mediator.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.      

Glucuronidation of 3-O-methylnoradrenaline, harmalol and some related compounds

1. The following compounds were glucuronidated in the presence of UDP-glucuronic acid and a microsomal preparation made from guinea-pig liver: (14)C-labelled 3-O-methyladrenaline, 3-O-methylnoradrenaline, 3-methoxytyramine and 4-hydroxy-3-methoxyphenethanol, as well as unlabelled harmalol and harmol. 2. [(14)C]Homovanillic (4-hydroxy-3-methoxyphenylacetic) acid was not a substrate for the microsomal glucuronyltransferase. 3. The K(m) values for harmalol and harmol were 0.69x10(-4)m and 0.50x10(-4)m respectively. 4. The K(m) values for UDP-glucuronic acid, in the presence of (14)C-labelled 3-O-methylnoradrenaline, harmalol and harmol as aglycones, were 0.57x10(-4)m, 0.44x10(-4)m and 2.20x10(-4)m respectively. 5. Mg(2+) added at 2.5-10mm activated glucuronyltransferase, with harmalol as substrate. Concentrations above 10mm inhibited the enzymic activity. 6. The overall, or net, transglucuronidating activity of microsomal preparations of the liver, with harmalol as substrate, was greatest for guinea pig, and very much lower for rabbit, mouse and rat.     

Uridine nucleotides in brain

—Two uridine nucleotides, UDPG and UDPGA, have been measured in brain by a procedure whereby they are extracted from tissue with subsequent utilization, either directly with (UDPGA) or after dehydrogenation (UDPG), in a glucuronidation reaction with harmol or harmalol as cosubstrate. The concentrations of UDPG and UDPGA in brains of a few species of animals examined are, respectively, 11.5–23.3 μmoles/100 g and 0.7–2.5 μmoles/100 g tissue. The ratios of UDPGA to UDPG range from 4% to 11%. The role and importance of these uridine nucleotides in brain are discussed.