The influence of glycosylation on the fate of renin expressed in Xenopus oocytes

It has been recently reported that, in Xenopus oocytes injected with the mRNA for human renin, this secretory renal glycoprotein acquires phosphomannosyl residues on its asparagine-linked oligosaccharide chains, remains intracellular and undergoes a proteolytic cleavage which removes the prosegment. To understand the influence of glycosylation on the fate of renin in Xenopus oocytes and whether it is specific for human renin, we have expressed human renin and mouse Ren1 renin, which are glycosylated at two and three selected asparagine residues, respectively, and mouse Ren2 renin, which is not glycosylated, in Xenopus oocytes. The majority of human and Ren1 renins remained intracellular and underwent proteolytic cleavage, whereas mouse Ren2 renin was secreted efficiently. When human and Ren1 renins were expressed in oocytes treated with tunicamycin, both were secreted efficiently. A mutant of human renin, which had amino-acid substitutions at both glycosylation sites, was also secreted efficiently, whereas that mutated at one of the two sites was not. These results indicate that the majority of all of the glycosylated renin molecules remain intracellular and undergo proteolytic cleavage, probably due to the acquisition of phosphomannosyl residues, and the human renin remains intracellular if it is only glycosylated at one of the two sites.     

Affinity labelling of endothelin receptor and characterization of solubilized endothelin-endothelin-receptor complex

Chick cardiac membranes were affinity labelled by cross-linking to membrane-bound 125I-endothelin-1 with disuccinimidyl tartarate. SDS/PAGE and autoradiographic analysis of the 125I-endothelin-1-labelled material in the presence or absence of 2-mercaptoethanol revealed one major labelled band, corresponding to a molecular mass of 53 kDa, whose appearance was dose-dependently inhibited by the addition of unlabelled endothelin-1 (1-100 nM). Subtracting the molecular mass of 125I-endothelin-1 and disuccinimidyl tartarate, the binding protein appeared to have a molecular mass of 50 kDa. To investigate further the molecular properties of endothelin receptor, the 125I-endothelin-1-endothelin-receptor complex was solubilized from chick cardiac membranes using the detergent digitonin. Sucrose gradient sedimentation of the solubilized complex indicated a sedimentation coefficient of 13 S, whereas the complex of (+)-[3H]PN200-110, a dihydropyridine derivative, and dihydropyridine-sensitive Ca2+ channels sedimented at 22 S. A monoclonal antibody raised against dihydropyridine-sensitive Ca2+ channels from the chick brain did not immunoprecipitate the 125I-endothelin-1-endothelin-receptor complex. These data suggest that endothelin receptor is clearly distinct from dihydropyridine-sensitive Ca2+ channels and endothelin has its own specific 50-kDa receptor.     

Carboxyphosphonoenolpyruvate phosphonomutase, a novel enzyme catalyzing C-P bond formation.

An enzyme catalyzing the formation of an unusual C-P bond that is involved in the biosynthesis of the antibiotic bialaphos (BA) was isolated from the cell extract of a mutant (NP71) of Streptomyces hygroscopicus SF1293. This enzyme, carboxyphosphonoenolpyruvate (CPEP) phosphonomutase, was first identified as a protein lacking in a mutant (NP213) defective in one of the steps in the pathway to BA. The first 30 residues of the amino terminus of this protein were identical to those predicted by the nucleotide sequence of the gene that restored BA production to NP213. The substrate of the enzyme, a P-carboxylated derivative of phosphoenolpyruvate named CPEP, was also isolated from the broth filtrate of NP213 as a new biosynthetic intermediate of BA. CPEP phosphonomutase catalyzes the rearrangement of the carboxyphosphono group of CPEP to form the C-P bond of phosphinopyruvate.     

Primate's p53 inhibits SV40 DNA replication in vitro

Previous reports indicated that rodent p53 inhibits simian virus 40 (SV40) DNA replication in vitro as well as in vivo while that from primate cells does not (1-4). Here we report the evidence that p53 of primate origin also inhibits SV40 DNA replication in vitro. p53-SV40 large tumor antigen (T antigen) complex purified from SV40 infected COS-1 cells had little replication activity and inhibited SV40 DNA replication in vitro. These results suggest that inhibition of SV40 DNA replication by p53 should be regarded as general property of the protein and does not determine the mode of species specific replication of SV40 DNA.     

Isolation of anti-endothelin receptor monoclonal antibodies for use in receptor characterization

Monoclonal antibodies reactive with endothelin (ET) receptors have been prepared by immunization of mice with rat lung membranes. Of four clones isolated, three clones preferentially recognized 32,000-dalton ET receptor and the other has a higher affinity for the 45,000-dalton receptor. The binding of 125I-ET-1 to detergent-solubilized ET receptors which were adsorbed to the antibodies was displaced by increasing concentrations of unlabeled ET isopeptides. These results demonstrate that the four clones specific for the receptor have the potential to be a useful tool in the characterization of ET receptors.     

NMR characteristics of intracellular K in the rat salivary gland: a 39K NMR study using double-quantum filtering

Intracellular K of the perfused rat mandibular salivary gland was measured by 39K NMR spectroscopy at 8.45 T. Multiple-quantum NMR arising from multiple-exponential decay was used to eliminate the resonance due to extracellular K in the perfused gland at 25 degrees C. The resonance due to intracellular K consisted of two Lorentzian signals stemming from the [spin 1/2 to -1/2] coherence (sharp resonance) and the [spin -1/2 to -3/2], [spin 3/2 to 1/2] coherences (broad resonance). The transverse relaxation time (T2) corresponding to the [spin 1/2 to -1/2] coherence was ca. 2.5 ms, and that corresponding to the [spin -1/2 to -3/2], [spin 3/2 to 1/2] coherences was ca. 0.4 ms. The relaxation time of the double-quantum coherence of rank 3 (originating from product operators like Ix2Iz) was determined to be ca. 0.2 ms. These results suggest the possibility of the presence of a single homogeneous population of intracellular K with a correlation time of ca. 2.5 x 10(-8) s and a quadrupolar coupling constant of ca. 1.4 MHz.     

Measurement of intracellular Na in the rat salivary gland: a 23Na-NMR study using double quantum filtering

23Na in the prefused rat mandibular salivary gland was measured by spin-echo double quantum filter 23Na-NMR spectroscopy at 8.45 T. Resonances due to the intracellular 23Na and the interstitial 23Na were observed in the perfused gland at 25 degrees C. The resonance due to intracellular 23Na consisted of two Lorentzian signals stemming from the [1/2 mean value of -1/2[ coherence (sharp resonance) and the [-1/2 mean value of -3/2[ and [3/2 mean value of 1/2[ coherences (broad resonance). The transverse relaxation rate constant corresponding to the [1/2 mean value of -1/2[ coherence was 95 +/- 4 s-1 and that corresponding to the [-1/2 mean value of -3/2[ and [3/2 mean value of 1/2[ coherences was 1360 +/- 75 s-1 (mean +/- S.E., n = 5). The resonance due to the interstitial 32Na had longer relaxation rate constants, and disappeared upon administration of dysprosium triethylenetetramine-N,N',N",N",N"'-hexaacetic acid.     

Isolation of temperature-sensitive cell cycle mutants from mouse FM3A cells. Characterization of mutants with special reference to DNA replication

A large number of mutants that are temperature sensitive (ts) for growth have been isolated from mouse mammary carcinoma FM3A cells by an improved selection method consisting of cell synchronization and short exposures to restrictive temperature. The improved method increased the efficiency of isolating DNA ts mutants, which showed a rapid decrease in DNA-synthesizing ability after temperature shift-up. Sixteen mutants isolated by this and other methods were selected for this study. Flow microfluorometric analysis of these mutants cultured at a nonpermissive temperature (39 degrees C) for 16 h indicated that five clones were arrested in the G1 to S phase of the cell cycle, six clones were in the S to G2 phase, and two clones were arrested in the G2 phase. The remaining three clones exhibited 8C DNA content after incubation at 39 degrees C for 28 h, indicating defects in mitosis or cytokinesis. These mutants were classified into 11 complementation groups. All the mutants except for those arrested in the G2 phase and those exhibiting defects in mitosis or cytokinesis showed a rapid decrease in DNA synthesis after temperature shift-up without a decrease in RNA and protein synthesis. The polyomavirus DNA cell-free replication system, which consists of polyomavirus large tumor antigen and mouse cell extracts, was used for further characterization of these DNA ts mutants. Among these ts mutants, only the tsFT20 strain, which contains heat-labile DNA polymerase alpha, was unable to support the polyomavirus DNA replication. Analysis by DNA fiber autoradiography revealed that DNA chain elongation rates of these DNA ts mutants were not changed and that the initiation of DNA replication at the origin of replicons was impaired in the mutant cells.     

Saccharomyces cerevisiae spheroplasts are sensitive to the action of diphtheria toxin.

Diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae but not the intact yeast cells. After 2 h of exposure to ca. 10(-7) M toxin, less than 1% of spheroplasts were able to regenerate into intact cells. The same high levels of toxin inhibited the rate of protein synthesis by more than 90% within 1 h, whereas RNA and DNA synthesis were not inhibited until 4 h or exposure. Both killing and protein synthesis inhibition were dependent on toxin concentration. The nature of the toxin-cell interaction was also studied by using fragments of intact toxin and mutant toxin proteins. Neither toxin fragment A nor CRM45 nor CRM197 affected spheroplasts, but CRM197 and ATP prevented the inhibitory action of intact toxin. These results suggest that toxin acts on S. cerevisiae spheroplasts in much the same manner as it acts on sensitive mammalian cells.     

Growth of mouse plasmacytoma cells in serum-free, hormone-supplemented medium: procedure for the determination of hormone and growth factor requirements for cell growth

The procedure of analyzing hormone and growth factor requirements for the growth of MPC-11 cells and of developing a serum-free medium for this cell line has been described. In this medium, MPC-11 cells grow as fast as in serum-supplemented medium, up to 50 generation. MPC-11 cells grown in serum-supplemented medium secrete IgG_2b and K light chain into the medium as they do in serum-containing medium.