Limited proteolysis reveals low-affinity binding of N-acetyl-L-glutamate to rat-liver carbamoyl-phosphate synthetase (ammonia)

Carbamoyl-phosphate synthetase was inactivated by elastase with first-order kinetics, and N-acetyl-L-glutamate speeded inactivation. From the dependence of the t1/2 value for inactivation on the concentration of acetylglutamate we estimate a Kd value for binding of the activator of 0.365 mM, which is approximately 600 times greater than in the presence of ATP, HCO3-, K+ and Mg2+. K+ and Mg2+ are not required for binding with low affinity, and in the absence of ATP they do not appear to increase the affinity for acetylglutamate. In the presence of acetylglutamate, mixtures of ATP, K+ and Mg2+ protect the enzyme from inactivation. ADP or AdoPP[NH]P partly replaced ATP in protecting the enzyme and thus binding of the nucleotide without further reaction is enough for protection. Two partial activities of the enzyme were inactivated by elastase to the same extent as the overall reaction, and thus elastase affects some property of the enzyme which is essential for catalysis. With other proteinases tested, inactivation was also accelerated by acetylglutamate and was slowed by mixtures of ATP, K+, Mg2+ and acetylglutamate, suggesting that changes in the accessibility of susceptible bonds are responsible for the changes in the degree of inactivation. It is concluded that elastase attacks at or close to the binding sites for ATP, and that exposure of the binding site for the ATP molecule that yields Pi (ATPA) upon binding of acetylglutamate causes the acceleration of the proteolytic inactivation.     

Salinity and germination of annual Melilotus from the Guadalquivir delta (SW Spain)

The germination response to NaCl treatments has been studied in Melilotus seed populations collected from saline and non-saline soils in the Guadalquivir delta. The rank orders for salt tolerance and seed weight were the same in the threeMelilotus species living in this area:Melilotus messanensis>M. segetalis>M. indica. Within the species, differences in germination response to salinity were found inM. indica (6 populations) andM. segetalis (8 populations). The relationship between salt tolerance during germination and salinity of maternal habitat is discussed.     

Leghemoglobin in Lupin Plants (Lupinus albus cv Multolupa)

Leghemoglobin was localized by immunogold techniques in nodules of Lupinus albus cv Multolupa inoculated with Bradyrhizobium sp. (Lupinus) strain ISLU 16. The protein localization was performed in nodules embedded in Spurr's and Araldite epoxy resins and Lowycryl K4M. A very good preservation of both the ultrastructure and antigenicity was obtained with the tissues embedded in Araldite following glutaraldehyde fixation and unpostfixed in osmium tetroxide. Lupin leghemoglobin is a stable and abundant protein which allows a conventional method to be safely used for localization of leghemoglobin. Labeling of leghemoglobin was specifically confined to the cytosol matrix and nuclei. Gold particles were never observed in the peribacteroidal spaces nor in the cytoplasmic organelles of the infected cells. Decrease of leghemoglobin was observed when the plants were grown with 10.7 micromolar and 21.4 micromolar of nitrate.     

Stages of B-lymphocyte differentiation in acute lymphoblastic leukemia

Blasts phenotype was determined in 61 children with the acute lymphoblastic leukemia. Non-T-cell acute lymphoblastic leukemia was diagnosed in 51 children. Stages of blasts differentiation were determined with the aid of monoclonal antibodies set using alkaline phosphatase-anti-alkaline phosphatase technique. Blasts in 50 patients belonged to B subpopulation confirmed by the presence of panB CD19 and CD22 antigens. Common antigen was seen in 76.5% of the examined patients with non-T-cell acute lymphoblastic leukemia. Cases of non-T-cell acute lymphoblastic leukemia were divided into 8 subgroups depending on the antigens of B-cells differentiation. An identification of pre-B subgroups of the acute lymphoblastic leukemia indicates heterogenicity of the acute lymphoblastic leukemias in childhood and enables their classification into groups corresponding to the early stages of lymphoblasts maturation.     

Various parameters of humoral and cellular immunity in children with iron deficiency

Serum IgA, IgM and IgG as well as percentage of lymphocytes T and B in peripheral blood were determined in 50 children aged between 6 months and 3 years with iron deficiency anaemia. A significant decrease in lymphocyte T number in these children was found in comparison with control group of healthy children. Lymphocyte T count positively correlated with serum iron concentration. Moreover, a decrease in serum IgA and IgG was found in children with iron deficit.     

Production of enterotoxin A by supposedly nonenterotoxigenic Staphylococcus aureus strains

The production of staphylococcal enterotoxins A (SEA) and B (SEB) was studied by inoculating six well-defined staphylococcal collection strains into cow's, goat's, or sheep's milk (individually or as a 50% mixture of cow's + goat's or cow's + sheep's), into brain heart infusion, and into a medium generally used to enhance the synthesis of enterotoxins (3+3 medium). Four of the strains used are considered to be SEB producers, another is considered an SEA producer, and the remaining strain is nonenterotoxigenic but produces large quantities of staphylococcal protein A. Staphylococcal protein A masked the results in most cases. Only one strain secreted exclusively SEB, while the other three SEB producers synthesized SEA in different amounts. We conclude that enterotoxin production depends on the natural substrate and may differ from the results obtained when the strain is grown on cellophane over agar to determine its toxigenicity.     

Posttranslational regulation of keratins: degradation of mouse and human keratins 18 and 8.

Human keratin 18 (K18) and keratin 8 (K8) and their mouse homologs, Endo B and Endo A, respectively, are expressed in adult mice primarily in a variety of simple epithelial cell types in which they are normally found in equal amounts within the intermediate filament cytoskeleton. Expression of K18 alone in mouse L cells or NIH 3T3 fibroblasts from either the gene or a cDNA expression vector results in K18 protein which is degraded relatively rapidly without the formation of filaments. A K8 cDNA containing all coding sequences was isolated and expressed in mouse fibroblasts either singly or in combination with K18. Immunoprecipitation of stably transfected L cells revealed that when K8 was expressed alone, it was degraded in a fashion similar to that seen previously for K18. However, expression of K8 in fibroblasts that also expressed K18 resulted in stabilization of both K18 and K8. Immunofluorescent staining revealed typical keratin filament organization in such cells. Thus, expression of a type I and a type II keratin was found to be both necessary and sufficient for formation of keratin filaments within fibroblasts. To determine whether a similar proteolytic system responsible for the degradation of K18 in fibroblasts also exists in simple epithelial cells which normally express a type I and a type II keratin, a mutant, truncated K18 protein missing the carboxy-terminal tail domain and a conserved region of the central, alpha-helical rod domain was expressed in mouse parietal endodermal cells. This resulted in destabilization of endogenous Endo A and Endo B and inhibition of the formation of typical keratin filament structures. Therefore, cells that normally express keratins contain a proteolytic system similar to that found in experimentally manipulated fibroblasts which degrades keratin proteins not found in their normal polymerized state.     

Incorporation of ionic channels from yeast plasma membranes into black lipid membranes.

Recently, patch-clamping of yeast protoplasts has revealed the presence of plasma membrane K+ channels (Gustin, M. C., B. Martinac, Y. Saimi, M. R. Culberston, and C. Kung. 1986. Science (Wash. DC). 233:1195-1197). In this work we show that fusion of purified plasma membranes into planar bilayers allows the study of the yeast channels. The main cationic conductances detected were of 64 and 116 pS, however, larger and smaller conductances have been observed. The two main conductances were sensitive to the K+ channels blockers tetraethylammonium (TEA+) and Ba2+. Bionic experiments indicated that both conductances were K+ selective.     

The use of functional analysis of the ribosome as a tool to determine archaebacterial phylogeny

Forty different antibiotics with diverse kingdom and functional specificities were used to measure the functional characteristics of the archaebacterial translation apparatus. The resulting inhibitory curves, which are characteristic of the cell-free system analyzed, were transformed into quantitative values that were used to cluster the different archaebacteria analyzed. This cluster resembles the phylogenetic tree generated by 16S rRNA sequence comparisons. These results strongly suggest that functional analysis of an appropriate evolutionary clock, such as the ribosome, is of intrinsic phylogenetic value. More importantly, they indicate that the study of the nexus between genotypic and phenotypic (functional) information may shed considerable light on the evolution of the protein synthetic machinery.