Hybridization of cloned Rhodopseudomonas capsulata photosynthesis genes with DNA from other photosynthetic bacteria

The homology of Rhodopseudomonas capsulata DNA segments carrying photosynthesis genes with sequences present in total DNA from certain other photosynthetic and non-photosynthetic bacterial species was determined by hybridization. R. capsulata DNA fragments that carry loci for production of peptide components of the photosynthetic reaction center and light-harvesting I antenna complex were found to hybridize to DNA from some photosynthetic species. However, fragments that carry carotenoid or bacteriochlorophyll biosynthesis genes showed either weak or undetectable heterospecific hybridization under the conditions employed.     

Immunochemical characterization of related families of glycoproteins in desmosomes

Using several biochemical approaches, we have characterized the relatedness of the various glycoprotein components of the bovine epidermal desomosome. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of purified epidermal desmosomes reveals 12 proteins, of which 8 are glycosylated. Analysis with monoclonal antibodies indicates that the 8 glycoproteins comprise 3 antigenically distinct protein families. Members of the highest molecular weight glycoprotein family (a triplet of Mr = 150,000) were not distinguishable by partial proteolytic peptide mapping. At least 6 different monoclonal antibodies have been identified that recognize unique antigenic determinants shared by these proteins. Members of a 97,000-118,000-dalton glycoprotein family (about 4 bands) generate very similar but not identical partial proteolytic peptide maps. At least 3 different monoclonal antibodies have been identified that recognize unique antigenic determinants shared by these proteins. A Mr = 22,000 glycoprotein is immunologically unrelated to either of the high molecular weight glycoprotein families. Lectin-binding profiles indicate that within each immunologically related family the glycoproteins are similar in their oligosaccharide composition. Some lectins distinguish among the families. These glycoproteins probably mediate the specific intercellular recognition and adhesive functions of the desmosome.     

A kinetic analysis of the effects of inhibitor-1 and inhibitor-2 on the activity of protein phosphatase-1

The steady-state interaction between protein phosphatase-1 and its two inhibitor proteins was studied in vitro at low enzyme concentrations where the assumptions of the Michaelis-Menten equation appeared to be valid. Under these conditions, and in the absence of divalent cations, inhibitor-1 behaved as a mixed inhibitor using phosphorylase alpha as a substrate, whereas inhibitor-2 was a competitive inhibitor. The results demonstrate that inhibitor-1 and inhibitor-2 do not interact with protein phosphatase-1 in an identical manner. Inhibitor-1 was only a substrate for protein phosphatase-1 in the presence of Mn2+, and its dephosphorylation was inhibited competitively by inhibitor-2 (Kis = 8 nM). Inhibitor-1 did not inhibit its own dephosphorylation in the presence of Mn2+. Its Km as a substrate (190 nM) was very much higher than its Ki as an inhibitor (1.5-7.5 nM). The results are consistent with a model in which a single binding site for inhibitor-1 is present on protein phosphatase-1, distinct from the binding site for phosphorylase alpha. It is envisaged that the binding of inhibitor-1 to this site not only inhibits the dephosphorylation of other substrates but permits access of its phosphothreonine to the same catalytic group(s) responsible for the dephosphorylation of other substrates. G-substrate, a protein phosphorylated exclusively on threonine residues, did not inhibit the dephosphorylation of phosphorylase alpha and its dephosphorylation was potently inhibited by inhibitor-1 or inhibitor-2. The role of the phosphothreonine residue in inhibitor-1 is discussed in the light of these results.     

l-Arginine and l-Canavanine Metabolism in Jack Bean, Canavalia ensiformis (L.) DC. and Soybean, Glycine max (L.) Merr

Studies have been conducted with the arginase (l-arginine amidinohydrolase, EC 3.5.3.1) of two legumes: jack bean, Canavalia ensiformis (L.) DC., a l-canavanine-containing plant and soybean, Glycine max, a canavanine-free species. Analyses of the arginase obtained from gradient-purified mitochondria of these legumes revealed that the arginine-dependent (ADA) and canavanine-dependent activities (CDA) were localized within this organelle.     

Indole-3-acetic Acid (IAA) and IAA Conjugates Applied to Bean Stem Sections: IAA Content and the Growth Response

High resolution growth recording techniques and reverse isotope dilution analysis were used to study the relationship between indole-3-acetic acid (IAA) concentration and curvature of excised bean (Phaseolus vulgaris L. cv Bush Burpee Stringless) first internode sections unilaterally treated with hormone. The maximum rate of curvature occurred rapidly (within 25 minutes) and was proportional to the log of the amount of applied IAA recovered in the tissue. The rate of curvature decreased after 30 minutes although little or no lateral migration of applied IAA occurred and tissue levels of IAA increased. The biologic activity of IAA-amino acid conjugates was found to be directly related to the amount of free IAA, resulting from their hydrolysis, which could be recovered from the tissue.     

Inhibition of virus-induced diabetes mellitus by interferon is influenced by the host strain

The diabetogenic variant of encephalomyocarditis virus (EMC-D) induces a diabetes-like syndrome in certain strains of mice. A study was done to determine if virus-induced diabetes could be prevented by interferon (IFN). It was found that the production of diabetes by EMC-D was blocked by either IFN beta or a variety of IFN-inducers in SWR/J, but not ICR Swiss mice. The replication of EMC-D in cell culture was inhibited by IFN beta. It is concluded that the response of pancreatic beta cells to the protective effect of IFN, is probably under genetic control.     

Studies of activating and nonactivating metal ion binding to yeast enolase

Measurements of binding of certain divalent cations to yeast apoenolase were made using a pH-meter, chromatography, a divalent cation electrode, and ultrafiltration. The binding of the activating metal ions Mg2+ and Co2+ and the nonactivator Ca2+ were studied as functions of the presence or absence of substrate/product, phosphate, and fluoride or level of Tb3+. The data suggest phosphate and fluoride increase Mg2+ binding but not Ca2+ binding. Substrate/product appears to increase Ca2+ binding as well as that of Mg2+ and Co2+. In the presence of substrate, Co2+ binding was 5-6 mol/mol dimer. In the absence of substrate/product, Tb3+ reduced Co2+ binding from 4 mol/mol to 2. These data are interpreted in terms of binding to "conformational," "catalytic" (substrate/product dependent), and "inhibitory" sites. Measurements of Tb3+ fluorescence quenching by Co2+ suggested that the distance between "conformational" sites on the two subunits was large, while the distance between "conformational" and "inhibitory" sites was ca. 17 +/- 4 A. Potentiometric titrations of apoenzyme with Ca2+ and Mg2+ showed that the metal ions produced the same proton release in the presence or absence of substrate/product. If phosphate and fluoride were present, then more protons were released if Ca2+ was the titrant rather than Mg2+, suggesting a difference in ionization state in the complex with the activating metal. Electron paramagnetic resonance studies of Co2+ binding to the various sites in the enzyme are presented. The Co2+ bound to all three sites appears to be high spin, consistent with a preponderance of oxyligands in an octahedral environment. Substrate, citrate, and a strongly binding substrate analogue strongly enhance the hyperfine structure of conformational Co2+. This is interpreted as the result of a change in interaction of an axial ligand to conformational Co2+ produced by carbon-3 of substrate or analogue.     

LARGE-SCALE ISOLATION AND FRACTIONATION OF ORGANS OF DROSOPHILA MELANOGASTER LARVAE

Methods for the mass isolation of diverse organs from small animals are described. They involve novel devices: a mechanical dissecting system, a centrifugal agitator for the separation of fibrillar from globular particles, and a settling chamber for the fractionation at unit gravity of particles with sedimentation velocities above the useful range for centrifugation. The application of these methods to the isolation of polytene and nonpolytene nuclei from Drosophila melanogaster larvae is described.     

An affinity column for renin

An affinity column for renin was prepared making use of the strong affinity of pepstatin for renin. Pepstatin is an N-acylated pentapeptide from $\text{Actinomycetes}$ with the following structure: isovaleryl-L-valyl-L-valyl-4-amino-3-hydroxy-6-methylheptanoyl-L-alanyl-4-amino-3-hydroxy-6-methylheptanoic acid. This peptide was coupled to aminoethylated polyacrylamide gel either directly with the water-soluble carbodiimide, 1-ethyl-3-(dimethylaminopropyl) carbodiimide, or through the N-hydroxy succinimide ester. Submaxillary renin was selectively retained by a small column of the gel and was eluted by a salt gradient to produce a highly pure material. This column was also effective for the purification of renal renin.