Effect of glycosylation on the mechanism of renaturation of invertase from yeast

N-Glycosylation occurs cotranslationally as soon as the growing polypeptide chain enters the endoplasmic reticulum, before the final native-like folded state is reached. We examined the role of the carbohydrate chains in the mechanism of protein folding. The in vitro folding and association of yeast invertase are used as an experimental system. External invertase contains approximately 50% carbohydrate, whereas cytoplasmic invertase is not glycosylated. The functional native state of both proteins is a homodimer. At pH greater than or equal to 6.5 and at protein concentrations below 3 micrograms/ml, the kinetics of reactivation and the final yields are similar for the two invertases. For both proteins, reactivation is a sequential reaction with a lag phase at the beginning. The nonglycosylated protein tends to aggregate during reactivation at low pH and at protein concentrations above 3 micrograms/ml. After separation of inactive material, the renatured protein is indistinguishable from the original native state by a number of physicochemical and functional criteria. The results suggest that the carbohydrate moiety does not affect the mechanism of folding and association of invertase. However, glycosylation improves the solubility of unfolded or partially folded invertase molecules and hence leads to a suppression of irreversible aggregation. Such a protective effect may also be important for the in vivo maturation of nascent glycosylated protein chains.     

Catalytic properties of chloroplast F1-ATPase modified at catalytic or noncatalytic sites by 2-azido adenine nucleotides

When heat-activated F1-ATPase from chloroplasts was repeatedly exposed to Mg2+ and 2-azido-ATP, followed by separation from medium nucleotides and photolysis, a total of two sites per enzyme, both catalytic and noncatalytic, were labeled. In a coupled assay with pyruvate kinase about half the activity was lost when one site per enzyme was modified. However, increased modification resulted in no further loss of activity. In contrast, methanol-sulfite activation of the enzyme showed a loss of most of the catalytic capacity when one site per enzyme was modified. Predominant labeling of either one catalytic or one noncatalytic site caused a loss of most of the activity in either assay. An indication that the enzyme modified at one site retained some catalytic activity was verified by measurement of the [18O]Pi species formed when [gamma-18O]ATP was hydrolyzed by partially derivatized enzyme. With either catalytic or noncatalytic site modification, the distributions of [18O]Pi species formed showed that the modified enzyme had different catalytic characteristics. An interpretation is that with modification by azido nucleotides at either catalytic or noncatalytic sites, capacity for rapid catalysis is largely lost but the remaining sites retain weak modified catalytic properties.     

The rate of uptake and efflux of phosphatidylcholine from human erythrocytes depends on the fatty acyl composition of the exchanging species

The rate of uptake of radioactive phosphatidylcholine molecules of different fatty acid composition in intact erythrocytes as facilitated by a phosphatidylcholine-specific transfer protein has been studied. When trace amounts of radiolabeled phosphatidylcholine molecules are present in donor vesicles consisting of egg phosphatidylcholine and cholesterol, the transfer of the radiolabeled species depends strongly on their fatty acyl composition: dipalmitoylphosphatidylcholine is transferred at the lowest rate, 1-saturated-2-unsaturated species are transferred faster and the highest rate is observed for dioleoyl phosphatidylcholine. Transfer of the various phosphatidylcholine molecules was measured furthermore using donor systems in which the bulk phosphatidylcholine was varied in its fatty acyl composition. Also in this type of experiment, the transfer protein preferentially stimulated transfer of unsaturated phosphatidylcholine molecules, especially from an environment containing more saturated molecules. Finally, the efflux of labeled phosphatidylcholine from intact erythrocytes to plasma in the absence of the phosphatidylcholine-specific transfer protein was studied and it became clear that in this case the nature of the effused molecules itself, rather than the composition of the bulk lipids, determined the effuse rates. An important conclusion to be drawn from these experiments is that radiolabeled phosphatidylcholine molecules, when used as markers for phospholipid exchange or transfer, should resemble in their fatty acid composition the composition of the bulk lipid in order to provide reliable data on rates and extents of the process studied.     

Fibronectin dependence of the contraction of collagen lattices by human skin fibroblasts

The role of fibronectin in the contraction of collagen lattices by human skin fibroblasts has been investigated. Incubation of lattice cultures in Dulbecco's modified Eagle's medium supplemented with increasing concentrations of non-dialysed or dialysed fetal calf serum demonstrated that the rate of contraction was dependent on non-dialysable serum components. The suppression of contraction observed when fibronectin was eliminated from serum, either by affinity chromatography on gelatin-agarose columns or by precipitation with anti-fibronectin antibodies, showed that fibronectin is critical for the contraction. When collagen lattices were incubated in a serum-free culture medium totally devoid of fibronectin, no contraction occurred. When fibronectin was added to this medium, their contraction was correlated with the concentration of fibronectin added. The contraction was inhibited by cycloheximide, tunicamycin, and monensin. These results demonstrate that the contraction of collagen lattices by human skin fibroblasts is dependent on fibronectin, and that other protein factors synthesized by the cells or contained in serum are also necessary.     

Synthesis and biological properties of antiparallel and parallel dimers of alpha-human atrial natriuretic peptide

To obtain antiparallel and parallel dimers of alpha-human atrial natriuretic peptide (alpha-hANP), two fully protected peptides I and II having the same amino acid sequence as alpha-hANP with different protective groups at the cysteinyl residues were synthesized, the former having Acm and Npys and the latter MeBzl and Acm. Equivalent amounts of peptides I and II were mixed and subjected to HF deprotection. Next, the first disulfide bond was linked between the remaining Npys group in I and the liberated SH group in II to form a monodisulfide dimer. The second disulfide bond was formed within the newly formed dimer between the remaining Acm groups by treatment with iodine, giving an antiparallel dimer. The parallel dimer of alpha-hANP was synthesized similarly starting from the protected peptide II. These dimers could be clearly segregated on HPLC. The retention time on HPLC of the antiparallel dimer was identical with that of natural beta-hANP. Both dimers showed biological activities as high as one third to one sixth of alpha-hANP in smooth muscle spasmolytic activity, and almost the same level of natriuretic activity as alpha-hANP at a high dose (10 nmol/kg) but about one fifth the activity at a low dose (1 nmol/kg). In these assay systems, the antiparallel dimer showed a slower onset and a tendency of longer duration than alpha-hANP.     

A monoclonal antibody that induces T cell aggregation reacts with vascular endothelial cells and placental trophoblasts

We have found that a mouse monoclonal antibody (alpha Leu-13) to a 16 kilodalton human lymphocyte surface antigen reacts with vascular endothelial cells as determined by immunoperoxidase staining of frozen tissue sections. In earlier studies, alpha Leu-13 was found to induce purified T cells to aggregate when added to cultures in nanogram concentrations. In the studies reported here, alpha Leu-13 stained vascular endothelial cells of arteries, capillaries, and veins in all organs examined from adults. It also reacted weakly with epithelial cells of proximal tubules of the kidney and with nonkeratinized basal epithelial cells of the cervix and esophagus. When a panel of tissues from a 14-wk-old fetus was examined, alpha Leu-13 was not found to react with endothelial cells of any specimen. However, it did stain medullary thymocytes and placental trophoblasts of this fetus. The implications of these findings to the possible function of the Leu-13 antigen in immune ontogeny are discussed.     

Folic acid and chromosome breakage. III. Types and frequencies of spontaneous chromosome aberrations in proliferating lymphocytes

The types and frequencies of spontaneous chromosome aberrations were studied in human lymphocytes cultured for 96 h in minimal essential medium (MEM) or MEM without folic acid (MEM-FA). In both media, the most frequent aberrations were chromatid gaps, isochromatid gaps and chromatid breaks. Chromosome (isochromatid) breaks and dicentrics were seen less frequently. Neither of these less frequent aberrations was seen in 4000 cells from MEM, but both were seen in 4000 cells from MEM-FA.     

Autocrine role of estrogens in the augmentation of luteinizing hormone receptor formation in cultured rat granulosa cells

The effects of estrogens on gonadotropin-stimulated luteinizing hormone (LH) receptor formation were examined in primary cultures of rat granulosa cells. Granulosa cells were cultured for 3 days with increasing concentrations of follicle-stimulating hormone (FSH) in the presence or absence of native and synthetic estrogens. Follicle-stimulating hormone stimulated LH receptor formation in a dose-dependent fashion, and estrogens enhanced the FSH-stimulated LH receptor content by decreasing the apparent ED50 of FSH. At 6.25 ng/ml FSH, the enhancement in LH receptor was estrogen dose dependent, with an ED50 value of about 3 X 10(-9) M for 17 beta-estradiol. The increased LH receptor content seen in cells treated with FSH and estrogen was correlated with increased cAMP production by these cells in response to LH stimulation. Time course studies revealed enhancement of FSH-stimulated LH receptor induction at 48 and 72 h of culture. Granulosa cells were also cultured with FSH for 2 days to induce functional LH receptors, then further cultured for 3 days with LH in the presence or absence of estrogens. At 30 ng/ml LH, increasing concentrations of estrogens maintained LH receptor content in a dose-dependent fashion, with their relative estrogenic potencies in keeping with reported binding affinities to estrogen receptors. An autocrine role of estrogens on LH receptor formation was further tested in granulosa cells treated with FSH and an aromatase substrate (androstenedione) to increase estrogen biosynthesis. Cotreatment with semipurified estrogen antibodies partially blocked the FSH stimulation of LH receptors, whereas nonimmune serum was ineffective. Also, inclusion of diethylstilbestrol prevented the inhibitory effect of the estrogen antibodies. Thus, local estrogens in ovarian follicles may play an autocrine role in granulosa cells to enhance LH receptor formation and to increase granulosa cell responsiveness to the LH surge, with subsequent ovulation and adequate corpus luteum formation.     

Sequential homologies between procarboxypeptidases A and B from porcine pancreas

Automated Edman degradation of monomeric procarboxypeptidases A and B from porcine pancreas shows that their N-terminal regions (from residue 1 to 34-37) present a high degree of sequential homology to each other as well as to other related procarboxypeptidases. Conformational predictions based on these sequences confirm their structural homology and indicate the probable existence of two beta-turns, one beta-chain and a long alpha-helix in them. On the other hand, tryptic peptide maps on a reverse-phase column indicate great sequential similarities (if not identity) between monomeric procarboxypeptidase A and the procarboxypeptidase A subunit isolated from its binary complex with proproteinase E.