Glycoproteins in bacterial membranes. In vivo labeling of the sugar portion of energy-transducing ATPase and a low-molecular-weight fraction fromMicrococcus lysodeikticus membranes

The energy-transducing ATPase and a low-molecular-weight fraction ofMicrococcus lysodeikticus membranes incorporated¹⁴C label fromd-[U-¹⁴C]glucose fed to the bacteria in synthetic medium. The specific radioactivity of the sugar portion of the ATPase and low-molecular-weight fraction was, respectively, 2.65 and 2.88 times that of their amino acids. Glucose and mannose in approximately equimolar amounts were identified as the main sugars of the glycoprotein ATPase, thus confirming previous structural studies. Glucose, galactose, and mannose (1:1:2) were identified as the main sugars of the low-molecular-weight glycopeptides. These results confirm and extend the notion that glycoprotein are constituents of prokaryotic membranes.     

Biosynthetic approach to the structure of F1-ATPase (BF1 factor) from Micrococcus lysodeikticus membranes: in vivo labeling of the polypeptides and study of their relationship

The F₁-ATPase or BF₁ factor was purified from Micrococcus lysodeikticus substrain B grown in a synthetic medium in the presence of tritiated amino acids. When analyzed in sodium dodecyl sulfate-7% polyacrylamide gels, the fresh purified preparation contained α, β, γ subunits (referred as the intrinsic subunits) and two other polypeptides (designated as X and component of relative mobility 1.0) whose status as subunits remains to be established. This overall polypeptide composition was similar to that of the F₁-ATPase isolated from the same strain grown in complex medium (J. Carreira, J. M. Andreu, M. Nieto, and E. Muñoz., 1976 Mol. Cell. Biochem.10, 67–76). The distribution of ³H-labeled amino acids into purified F₁-ATPase and its constituent polypeptides under different stages of growth was used to investigate the biosynthetic relationship between the different polypeptides. The incorporation of amino acids into purified BF₁ factor was slower than that of cytoplasmic and other membrane proteins. In isotope-dilution and chase experiments, F₁-ATPase showed one of the slowest rates of decay of the incorporated label. These results point out that F₁-ATPase of M. lysodeikticus undergoes slower turnover than the overall cytoplasmic and membrane proteins. Pulse and chase experiments allowed us to conclude that the α, β, γ subunits and the components of relative mobility 1.0 are independent with differences in their turnover and therefore do not bear any apparent relation as precursors-products. The two major subunits represent seemingly the “core” of ATPase, the β subunit behaving like the most stable component. On the other hand, the γ subunit appears to be synthesized independently from this α + β complex.     

Morphological changes in strains of Aspergillus flavus link ex fries and Aspergillus parasiticus speare related with aflatoxin production

Two strains ofAspergillus flavus Linkex Fr. and two strains ofA. parasiticus Speare were cultured on crushed moist wheat (Triticum durum var. Pané no. 247) for aflatoxin production studies in correlation with morphological changes. The toxicogenic strains were adapted to the substratum by means of successive transfers at regular intervals (72 h.)The amount aflatoxins synthesized by the toxicogenic strains decreased gradually after succesive subculturing. The decrease was accompanied by marked morphological changes. One of the strains studied,A. flavus NRRL 3251, lost completly the capacity of aflatoxin synthesis after several subcultures, presenting at the same time strong morphological variations.A. flavus CBS 120.62 also lost its toxicogenicity after six subcultures.     

Retinoblastoma,gross internal malformations,and deletion 13q14→q31

Summary A male patient with an interstitial deletion 13q14q31 is described. Our necropsy findings included a left retinoblastoma and several gross internal malformations. In this paper we reaffirm that band 13q14 is involved in cases of retinoblastoma and we propose, after studying accompanying cases of total or partial long arm trisomies 13, that the loss of specific 13q bands, from 13q14 to 13q31 is responsible for the congenital defects we are describing.     

Immunochemistry of membrane F1-Adenosine triphosphatase fromMicrococcus lysodeikticus. Role of the alpha subunit

The membrane-bound ATPase activity from two substrains ofMicrococcus lysodeikticus, designated as A and B, was inhibited by antibodies raised against the two forms of purified F₁-ATPase. Form B of the enzyme, which behaved as a poorer immunogen than form A, also showed less reactivity as an antigen, independent of the physical state of the F₁-ATPase form. Antibodies were raised against the two major subunits ( and ) isolated fromM. lysodeikticus F₁-ATPase form A, which was the most stable form of the enzyme. Anti-(-subunit) serum strongly inhibited the ATPase activity of membrane-bound ATPase but showed little inhibition of the purified, soluble F₁-ATPase. The anti-(-subunit) serum inhibited the soluble F₁-ATPase, but to a lesser extent than the membrane-bound enzyme. In any event, the effect of anti- antibodies on the membrane-bound ATPase was smaller than that of anti- antibodies. It was postulated that the subunit ofM. lysodeikticus F₁-ATPase plays an essential and regulatory role in the expression of the immunochemical properties of the protein.     

Dipole moments of random coil polypeptides

The Rotational Isomeric States model is applied to calculate dipole moments of polypeptides of the twenty natural α-amino acids in the random coil state. Dipole moments of each repeat unit (μ_i), are evaluated using a quantum mechanics procedure. Dipole moment ratios ($\text{D}{}_{\mathrm{x}}\text{=}\text{〈μ}{}^2\text{〉}\text{x}\text{μ}{}_{\mathrm{i}}{}^2\text{,}\text{x = number of repeat units}$) of homopolypeptides are calculated and extrapolated to x →?. With a few exceptions, D_? = 0.36 ± 0.1. Ten actual proteins and three enzymes are also studied; their dipole ratios (D_x′ =〈μ〉/x) range from 7.34 to 10.57 in 10^?59 C² m² (6.6–9.5 D²). Diffferences in the values of D_x′ are due mainly to the different contributions, μ_i, of the amino acid residues contained in each polymer, whereas the sequence of amino acids has a very minor effect.     

Monoclonal antibodies specific for the core protein of the beta-subunit of the gastric proton pump (H+/K+ ATPase). An autoantigen targetted in pernicious anaemia.

The gastric H+/K(+)-transporting adenosine triphosphatase (H+/K+ ATPase) (proton pump) consists of a catalytic alpha-subunit and a recently proposed 60-90-kDa glycoprotein beta-subunit. Using dog gastric membranes as the antigen, we have produced two murine monoclonal antibodies, 4F11 (IgG1) and 3A6 (IgA), which are specific for the 60-90-kDa glycoprotein. The monoclonal antibodies (1) specifically stained the cytoplasm of unfixed and formalin-fixed dog gastric parietal cells; (2) specifically reacted by ELISA with gastric tubulovesicular membranes; (3) recognised epitopes located on the luminal face of parietal cell tubulovesicular membranes, the site of the proton pump, by immunogold electron microscopy; (4) immunoblotted a 60-90-kDa molecule from tubulovesicular membranes and a 35-kDa component from peptide N-glycosidase-F-treated membrane extracts; (5) immunoblotted the 60-90-kDa parietal cell autoantigen associated with autoimmune gastritis and pernicious anemia, purified by chromatography on parietal cell autoantibody- or tomato-lectin-Sepharose 4B affinity columns, and the 35-kDa protein core of this autoantigen; this autoantigen has amino acid sequence similarity to the beta-subunit of the related Na+/K(+)-transporting adenosine triphosphatase (Na+/K+ ATPase) [Toh et al. (1990) Proc. Natl Acad. Sci. 87, 6418-6422]; (6) co-precipitated a molecule of 95 kDa with the 60-90-kDa molecule from 125I-labelled detergent extracts of dog tubulovesicular membranes; and (7) co-purified the catalytic alpha-subunit of the H+/K+ ATPase with the 60-90-kDa molecule by immunoaffinity chromatography of tubulovesicular membrane extracts on a monoclonal antibody 3A6-Sepharose 4B column, indicating a physical association between the two molecules. These results provide further evidence that the 60-90-kDa glycoprotein is the beta-subunit of the gastric H+/K+ ATPase. We conclude that the monoclonal antibodies specifically recognise luminal epitopes on the 35-kDa core protein of the 60-90-kDa beta-subunit of the gastric proton pump, a major target molecule in autoimmune gastritis and pernicious anaemia. These monoclonal antibodies will be valuable probes to study the structure and function of this associated beta-subunit, as well as the ontogeny of the gastric proton pump.     

A limnological reconnaissance of Lake Tebenquiche,Salar de Atacama,Chile

A number of expeditions to the area of Salar de Atacama, Chile, 68° 15'W, 20° 30'S, have involved studies of the biological and chemical features of Lake Tebenquiche, situated in the interior of the salar. Chemically, Tebenquiche is hypersaline, with practically anoxic waters dominated by sodium and chloride ions but with high concentrations of sulphate also. The lake is surrounded and invaded by macrophytes, dominated by Scirpus olmeyi and Juncus, which provide organic material for the formation of bacterial mats. The fauna of limnetic crustaceans is almost exclusively of Artemia salina. The most important genera of bacteria are: Marinomonas, Halobacterium, Acinetobacter and the sulphur reductors Vibrio and Bacillus. The Cyanobacteria are represented exclusively by Oscillatoria.     

Deoxyribonuclease activities in Myxococcus coralloides D

Myxococcus coralloides D produced cell-bound deoxyribonucleases (DNases) during the exponential phase of growth in liquid medium. DNase activity was much higher than that detected in other myxobacterial strains and was fractionated into three different peaks by filtration through Sephadex G-200. The DNases were named G, M and P. The optimum temperatures were 37 degrees C, 33 degrees C and 25 degrees C respectively, although high activities were recorded over the temperature range 20-45 degrees C. The pH range of high activity was between 6.0 and 9.0, with an optimum for each DNase at 8.0. DNases M and P were strongly inhibited by low concentrations of NaCl, but activity of DNase G was less affected by NaCl. The three activities required divalent metal ions as cofactors (especially Mg2+ and Mn2+); however, other metal ions (Fe2+, Ni2+, Zn2+) were inhibitors. The molecular weights were estimated by gel filtration chromatography and SDS-PAGE as 44 kDa (DNase G), 49 kDa (DNase M) and 39 kDa (DNase P).