Sequence interrelationships of the subunits of vicilin from pea seeds

Serological studies and comparison of N-terminal amino acid sequences with the amino acid sequence deduced from a cDNA clone have been used to establish the sequence relationships between the subunits of the pea seed storage protein, vicilin. Subunits smaller than M_r～50 000 (i.e., M_r 34 000, 30 000, 25 000, 18 000, 14 000, 13 000 and 12 000) show extensive homology with molecules within M_r～50 000 group. Both the sequencing and serological data confirm earlier evidence from studies on vicilin synthesisin vivo andin vitro which indicated that the vicilin subunits smaller than M_r～50 000 arose by endoproteolytic cleavage of parent molecules within the M_r～50 000 group. Cleavage in different M_r 50 000 parent molecules containing either one or both of two susceptible processing sites accounts for the formation of all the vicilin subunits smaller than M_r～50 000, with the possible exception of the M_r34 000 polypeptide. The position of these sites in the putative parents were defined by reference to a complete amino acid sequence deduced from the sequence of DNA complementary to mRNA for one member of the M_r～50 000 group.     

Structure and function in the excretory systems of some terrestrial prosobranch snails (Cyclophoridae)

The excretory systems of terrestrial prosobranch snails of the family Cyclophoridae, collected in Jamaica, Costa Rica and South Africa, have been examined physiologically and as regards their gross and fine structure. The process of urine formation commences in the heart, where fluid is filtered across the wall of the ventricle. Filtration through the auricular wall is believed to be negligible. The kidney, which contains three types of cell, modifies the composition of the filtrate. One type of resorptive cell, characterized by basal infoldings associated with mitochondria, takes up salts. Another type, with basal subcellular spaces, may be responsible for taking up water. The third type of cell is secretory, producing concretions of uric acid and phospholipid which are liberated into the kidney lumen when the cell degenerates.
The rate and mechanism of urine production have been investigated using injections of inulin. The filtration rate at 25°C is 0.5 μl/g/min, and in 100% R.H. the average rate of urine production is 0–39 μl/g/min.
An accessory excretory organ has been developed from the hypobranchial gland of aquatic forms. It is composed of groups of subepithelial tubular glands opening into the mantle cavity by one or a series of pores, and secreting purines, phospholipids and mucus. There is evidence that this organ becomes progressively more complex in forms occupying drier habitats.
The systems of excretion and osmoregulation in the Cyclophoridae are considered to be very similar to those in their aquatic relatives, the Viviparidae and Ampullariidae. Certainly the cyclophorids are not as well adapted to a terrestrial life as are the Pulmonata, and in many respects they may be considered "aquatic" snails living on land.     

Inhibition by local anaesthetics of adenine nucleotide translocation in rat liver mitochondria

1. The mechanism of adenine nucleotide translocation in mitochondria isolated from rat liver was further examined by using the local anaesthetics procaine, butacaine, nupercaine and tetracaine as perturbators of lipid-protein interactions. Each of these compounds inhibited translocation of ADP and of ATP; butacaine was the most effective with 50% inhibition occurring at 30mum for 200mum-ATP and at 10mum for 200mum-ADP. The degree of inhibition by butacaine of both adenine nucleotides was dependent on the concentration of adenine nucleotide present; with low concentrations of adenine nucleotide, low concentrations of butacaine-stimulated translocation, but at high concentrations (greater than 50mum) low concentrations of butacaine inhibited translocation. Butacaine increased the affinity of the translocase for ATP to a value which approached that of ADP. 2. Higher concentrations of nupercaine and of tetracaine were required to inhibit translocation of both nucleotides; 50% inhibition of ATP translocation occurred at concentrations of 0.5mm and 0.8mm of these compounds respectively. The pattern of inhibition of ADP translocation by nupercaine and tetracaine was more complex than that of ATP; at very low concentrations (less than 250mum) inhibition ensued, followed by a return to almost original rates at 1mm. At higher concentrations inhibition of ADP translocation resulted. 3. That portion of ATP translocation stimulated by Ca(2+) was preferentially inhibited by each of the local anaesthetics tested. In contrast, inhibition by the anaesthetics of ADP translocation was prevented by low concentrations of Ca(2+). 4. The data provide further support for our hypothesis that lipid-protein interactions are important determinants in the activity of the adenine nucleotide translocase in mitochondria.     

Fate of corticotrophins in an isolated adrenal-cell bioassay and decrease of peptide breakdown by cell purification

1. The fate of corticotrophins in a trypsin-dispersed rat adrenal-cell assay system was investigated with a view to establishing whether differences in the rate of inactivation might contribute to potency differences observed between analogues. 2. Corticotrophin-(1-24)-tetracosapeptide and to a lesser extent synthetic 1-39 corticotrophins were found to be inactivated during incubation with cell suspension. 3. Peptide fragments were isolated by using [[(3)H(2)]Tyr(23)]corticotrophin-(1-24)- tetracosapeptide as a marker. The fragments indicate a peptidase with a predominantly tryptic specificity. 4. The peptidase is present in the extracellular fluid and is released from cells when they are damaged. 5. Cells were fractionated on an albumin gradient. Cells from the zona fasciculata and the zona intermedia or reticularis were present in fractions which produced fluorogenic steroids in response to corticotrophin. 6. Purification of the cells by centrifugation through albumin decreased degradation by peptidases, so that if the assay is carried out with a dilute suspension of purified cells peptide breakdown should not affect the observed potencies of adrenocorticotrophin analogues. 7. No binding of [[(3)H(2)]Tyr(23)]corticotrophin-(1-24)- tetracosapeptide to cells could be detected at low concentrations of the peptide. This indicated that less than 120 receptors/cell are occupied during stimulation by a dose that would elicit approx. 80% of the maximal response.     

Studies on partially reduced mammalian cytochrome oxidase. Reactions with carbon monoxide and oxygen

A number of methods were used to prepare a species of mammalian cytochrome oxidase (EC 1.9.3.1, ferrocytochrome c-oxygen oxidoreductase) in which only cytochrome a(3) is reduced and in combination with CO. The kinetics of CO binding by cytochrome a(3) (2+) in this species is significantly different from that exhibited by cytochrome a(3) (2+) in the fully reduced enzyme. The second-order rate constant for combination was 5x10(4)m(-1).s(-1) and the ;off' constant was 3x10(-2)s(-1). The kinetic difference spectra cytochrome a(3) (2+)-cytochrome a(3) (2+)-CO reveal further differences between the mixed-valence and the fully reduced enzyme. The reaction between cytochrome a(3) (2+) and oxygen in the mixed-valence species was followed in flow-flash experiments and reveals a fast, oxygen-dependent (8x10(7)m(-1).s(-1) at low oxygen) rate followed by a slow process, whose rate is independent of oxygen but whose amplitude is dependent on [O(2)]. The fast oxygen-dependent reaction yields as the first product the so-called ;oxygenated' enzyme. We conclude from these experiments that the ligand-binding behaviour of cytochrome a(3) depends on the redox state of its partners, a fact which represents clear evidence for site-site interaction in this enzyme. The fact that oxygen reacts rapidly with this enzyme species in which only one component, namely cytochrome a(3), is reduced represents clear and unequivocal evidence that this is indeed the O(2)-binding site in cytochrome oxidase and may indicate that reduction of oxygen can proceed via single electron steps.     

Production and utilization of acetate in mammals

1. In an attempt to define the importance of acetate as a metabolic precursor, the activities of acetyl-CoA synthetase (EC 6.2.1.1) and acetyl-CoA hydrolase (Ec 3.1.2.1) were assayed in tissues from rats and sheep. In addition, the concentrations of acetate in blood and liver were measured, as well as the rates of acetate production by tissue slices and mitochondrial fractions of these tissues. 2. Acetyl-CoA synthetase occurs at high activities in heart and kidney cortex of both species as well as in rat liver and the sheep masseter muscle. The enzyme is mostly in the cytosol fraction of liver, whereas it is associated with the mitochondrial fraction in heart tissue. Both mitochondrial and cytosol activities have a K(m) for acetate of 0.3mm. Acetyl-CoA synthetase activity in liver was not altered by changes in diet, age or alloxan-diabetes. 3. Acetyl-CoA hydrolase is widely distributed in rat and sheep tissues, the highest activity being found in liver. Essentially all of the activity in liver and heart is localized in the mitochondrial fraction. Hepatic acetyl-CoA hydrolase activity is increased by starvation in rats and sheep and during the suckling period in young rats. 4. The concentrations of acetate in blood are decreased by starvation and increased by alloxan-diabetes in both species. The uptake of acetate by the sheep hind limb is proportional to the arterial concentration of acetate, except in alloxan-treated animals, where uptake is impaired. 5. Acetate is produced by liver and heart slices and also by heart mitochondrial fractions that are incubated with either pyruvate or palmitoyl-(-)-carnitine. Liver mitochondrial fractions do not form acetate from either substrate but instead convert acetate into acetoacetate. 6. We propose that acetate in the blood of rats or starved sheep is derived from the hydrolysis of acetyl-CoA. Release of acetate from tissues would occur under conditions when the function of the tricarboxylic acid cycle is restricted, so that the circulating acetate serves to redistribute oxidizable substrate throughout the body. This function is analogous to that served by ketone bodies.     

Isolation and structure of nocobactin NA, a lipid-soluble iron-binding compound from Nocardia asteroides

Nocobactin NA, a lipid-soluble iron-chelating product with an unusual and characteristic u.v.-absorption spectrum, was isolated from Nocardia asteroides grown under conditions of iron deficiency. Its structure was determined by physical methods and by synthesis of one of its degradation products. Nocobactin NA was obtained as a homologous mixture of compounds with side chains of differing length, and resembles mycobactin M in structure except that it has an oxazole ring in place of an oxazoline ring, and the side chains in the cobactin fragment are considerably shorter.     

Branched-chain glycosyl α-amino acids : Part II. Synthesis of 2-D- and 2-L-(3-deoxy-1,2:5,6-di-O-isopropylidene-α-D-glucofuranos-3-yl)glycine. Analogs of the sugar moiety of the polyoxins

Stereospecific hydroxylation of 3-deoxy-1,2:5,6-di-O-isopropylidene-3-C-trans-and 3-C-cis-(methoxycarbonylmethylene)-α-D-ribo-hexofuranose (2 and 3, respectively), with potassium permanganate in pyridine afforded 3-C-[S- and R-hydroxy-(methoxycarbonyl)methyl]-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose, (6 and 7, respectively), in a combined yield, after chromatography, of 43%. Selective formation of monomethanesulfonates (9a and 10a) and p-toluenesulfonates (9b and 10b), followed by treatment with sodium azide and reduction of the azide, afforded the methyl 2-D-(and 2-L-)(3-deoxy-1,2:5,6-di-O-isopropylidene-α-D-glucofuranos-3-yl)-glycinates (12a and 13a, respectively). Basic hydrolysis of the latter compounds yielded 2-D- and 2-L-(3-deoxy-1,2:5,6-di-O-isopropylidene-α-D-glucofuranos-3-yl)glycine (12b and 13b, respectively). The structures of the glycosyl amino acids were correlated with that of L-alanine by circular dichroism.     

Effect of Lytic Enzymes of Acanthamoeba castellanii on Bacterial Cell Walls

Extracts of Acanthamoeba castellanii (Neff) contain alpha- and beta-glucosidase, beta-galactosidase, beta-N-acetylglucosaminidase, amylase, and peptidase. All of these activities are optimal between pH 3 and 4. These extracts also were found to clarify suspensions of cell walls from nine different gram-positive bacteria, including Micrococcus lysodeikticus. The pH optimum for the lytic activity was between 3 and 4. The extent of lysis of the various cell walls did not correlate with the release of free amino groups and of free N-acetylated sugars from the walls during digestion with these extracts. Suspensions of cell walls of Escherichia coli (a gram-negative bacterium), Cordiceps militaris (a fungus), and Acanthamoeba cysts, as well as of colloidal chitin, were not clarified by incubation with these extracts, although reducing sugars were released from each of these materials. Exhaustive digestion of M. lysodeikticus walls by lysozyme released no free N-acetylglucosamine. The products of exhaustive digestion of this cell wall with Acanthamoeba extracts were free N-acetylglucosamine, free N-acetylmuramic acid, glycine, alanine, glutamic acid, lysine, and N-acetylmuramic acid peptide fragments. These results suggest that the amoeba extracts contain endo- and exo-hexosaminidases, in addition to beta-hexosaminidase and peptide hydrolases.