Structural transition of alpha 1-antitrypsin by a peptide sequentially similar to beta-strand s4A

Crystal structure studies have shown that cleaved and intact serpins differ essentially in the topology of beta-sheet A. This is five-stranded in the intact molecules and six-stranded after cleavage by insertion of strand s4A whose C-terminus has become free [L?bermann, H., Tokuoka, R., Deisenhofer, J. & Huber, R. (1984) J. Mol. Biol. 177, 531-556; Wright, T. H., Qian, H. X. & Huber, R. (1990) J. Mol. Biol. 213, 513-528]. The structural transition is accompanied by changes in spectral properties and an increase in thermal stability. We show here that an N alpha-acetyl-tetradecapeptide with the amino acid sequence of strand s4A, residues 345-358 of human alpha 1-antitrypsin, associates with intact alpha 1-antitrypsin and forms a stoichiometric complex with properties very similar to cleaved alpha 1-antitrypsin. Complex generation has the characteristics of a folding process.     

Expression of voltage-gated K+ channels in insulin-producing cells. Analysis by polymerase chain reaction.

We have used the polymerase chain reaction (PCR) with primers against the S5 and S6 regions of voltage-gated K+ channels to identify 8 different specific amplification products using poly(A)+ RNA isolated from islets of Langerhans from obese hyperglycemic (ob/ob) mice and from the two insulin-producing cell lines HIT T15 and RINm5F. Sequence analysis suggests that they derive from mRNAs coding for a family of voltage-gated K+ channels; 5 of these have been recently identified in mammalian brain and 3 are novel. These hybridize in classes to different mRNAs which distribute differently to a number of tissues and cell lines including insulin-producing cells.     

Construction by site-directed mutagenesis of a 39-kilodalton mosquitocidal protein similar to the larva-processed toxin of Bacillus sphaericus 2362.

After ingestion of the parasporal crystals of Bacillus sphaericus, mosquito larvae process the 42-kilodalton (kDa) toxin to a protein of 39 kDa, which has an increased toxicity (A. H. Broadwell and P. Baumann, Appl. Environ. Microbiol. 53:1333-1337, 1987). A similar activation is performed by trypsin and chymotrypsin. Using site-directed mutagenesis, we have constructed derivatives of the 42-kDa toxin with a deletion of 10 amino acids at the N terminus and deletions of 7, 17, or 20 amino acids at the C terminus. Toxicity for mosquito larvae was retained upon deletion of 7 or 17 amino acids but was lost upon deletion of 20 amino acids. Evidence is presented indicating that the protein containing deletions of 10 amino acids at the N terminus and 17 amino acids at the C terminus (corresponding to potential chymotrypsin cleavage sites) is similar to the 39-kDa protein produced in mosquito larvae or by digestion with chymotrypsin. Digestion with trypsin appears to generate a protein lacking 16 or 19 amino acids from the N terminus and 7 amino acids from the C terminus. As is the case with the recombinant-made 42-kDa protein, toxicity of its derivatives is dependent on the presence of a 51-kDa protein which is a component of the parasporal crystal of B. sphaericus 2362.     

Comparative gas phase and pulsed liquid phase sequencing on a modified Applied Biosystems 477A sequencer

A simple and inexpensive modification of the Applied Biosystems 477A sequencer, to run in the pulsed liquid-phase and in the gas-phase mode of the Edman chemistry, is described. This modification is especially useful for sequencing samples on polyvinylidene difluoride (PVDF) membranes. Additional carriers are required if a sample on a PVDF membrane is sequenced with the pulsed liquid-phase degradation program of the 477A. In the gas-phase mode no such carriers are needed. This eliminates time-consuming preconditioning sequencer cycles and reduces the sequencer background. In addition, initial coupling yields in the gas-phase mode exceeded those in the pulsed liquid-phase mode, whereas the average repetitive yields were similar. Samples spotted onto glass fiber filters pretreated with polybrene and samples spotted or electroblotted onto PVDF membranes were examined. A number of advantages of the gas-phase mode are presented.     

Modifications of ganglioside composition in peripheral nerve of myelin deficient trembler mutant mouse

Ganglioside distribution was studied in peripheral nerves of normal controls and those of Trembler mutant mouse with defect in Schwann cell differentiation and myelination. Neuraminic acid content was considerably decreased in the mutant. Ganglioside distribution as evaluated by densitometry of resorcinol positive spots on thin-layer chromatography revealed a major peak for GDl_a in normal controls. In the mutant, the relative proportion was modified with qualitative modifications in the GDl_a area and a tremendous increase in GM₃ content. The relation with the intense Schwann cell proliferation observed in the mutant is discussed.     

Bead rings at the endoplasmic reticulum-golgi complex boundary: morphological changes accompanying inihibition of intracellular transport of secretory proteins in arthropod fat body tissue

Golgi complex beads are 10-nm particles arranged in rings on the smooth surface of rough endoplasmic reticulum (ER) makind the forming face of the Golgi complex (GC). In arthropod cells they stain specifically with bismuth. Their morphology has been studied after treatment with reagents known to interfere with GC function. Inhibitors of oxidative phosphorylation (antimycin A, cyanide, and anoxia), but not an inhibitor of glycolysis (iodoacetate), both cause the bead rings to collapse and the GC saccules to round up, and inhibit transition vesicle (TV) formation. Cycloheximide blocks protein synthesis on ribosomes but does not stop TV formation or disrupt bead rings, even after prolonged treatment (6 h) to allow emptying of the rough ER cisternae. Thus the collapse of bead rings is not attributable to inhibition of protein synthesis, and the ring structure of beads does not require continued protein synthesis and secretion for its maintenance. Valinomycin has effects on the GC similar to those of antimycin A, but {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, monensin, and lasalocid do not affect bead ring structure or TV formation. These results are consistent with valinomycin’s secondarily uncoupling mitochondria, which collapses bead rings and prevents TV formation. Thus inhibitors of oxidative phosphorylation do not influence the beads through cation movement. Because mononsin and lasalocid block secretion at the level of the condensing vacuoles, bead rings are not influenced by blocks in secretion distal to them or by the backup of secretory material. These experiments are consistent with inhibitors of oxidative phosphorylation collapsing bead rings by decreasing intracellular ATP. The concomitant block to TV formation and the collapse of bead rings suggests that integrity of the bead rings is essential for the transport of secretory material from the rough ER to the GC.     

Study of relationship among species ofVibrio,Photobacterium, and terrestrial enterobacteria by an immunological comparison of glutamine synthetase and superoxide dismutase

The amino acid sequence divergence of glutamine synthetase (GS) from species ofVibrio, Photobacterium, Aeromonas, Escherichia, Salmonella, Citrobacter, Enterobacter, Serratia, Proteus, Erwinia, Xenorhabdus, andPlesiomonas was determined by quantitative microcomplement fixation, using antisera to GS fromVibrio alginolyticus andEscherichia coli. A similar study was performed with superoxide dismutase (SOD), using antiserum to the enzyme fromV. alginolyticus. A comparison of the results for GS and SOD, relative to the enzymes fromV. alginolyticus, as well as a comparison of these data with the results of previous ribosomal RNA (rRNA)/DNA homology studies indicated a high degree of congruence (correlation coefficients≥0.9). The results with both enzymes suggested four major groupings among these genera: (i)Vibrio, (ii)Photobacterium, (iii)Aeromonas, (iv) a large and heterogeneous group which included the peritrichously flagellated terrestrial enterobacteria.