Folding changes in membrane-inserted diphtheria toxin that may play important roles in its translocation.

Diphtheria toxin membrane penetration is triggered by the low pH within the endosome lumen. Subsequent exposure to the neutral pH of the cytoplasm is believed to aid in translocation of the catalytic A domain of the toxin into the cytoplasm. To understand the effects of low pH and subsequent exposure to neutral pH on translocation, we studied toxin conformation in solution and in toxin inserted in model membranes. Two conformations were found at low pH. One form, L', predominates below 25-30 degrees C, and the other, L", predominates above 25-30 degrees C and is formed from the L' state by an unfolding event. Both forms are hydrophobic and penetrate deeply into membranes. After pH neutralization, the L' and L' conformations give rise to two new conformations, R' and R', respectively. The R' and R" conformations differ from each other in that in the R' state the A domain remains folded, whereas in the R" state the A domain is unfolded. This is confirmed by the finding that only the R' state possesses the capacity to bind and hydrolyze NAD+. It is also supported by the finding that the R' state can also be formed by thermal unfolding of the R' state. The R conformations differ from the low-pH L conformations in that although they remain largely membrane-inserted, it appears that a large portion of the toxin is no longer in contact with the hydrophobic core of the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)     

13C NMR relaxation and conformational flexibility of the deoxyribose ring.

13C T1's and NOE's have been measured for all protonated carbons of 2'-deoxy-D-ribose (2'-d-ribose), 2'-deoxyadenosine-5'-monophosphate (5'-dAMP), thymidine-3'-monophosphate (3'-TMP) and thymidine-5'-monophosphate (5'-TMP) in D2O solutions. In all of the deoxy sugars examined, NT1 values for C-2' are significantly larger than the values for the remaining carbons. This result is interpreted in terms of rapid puckering motion of C-2'. By contrast, NT1 values measured in ribose are found to be equal, within experimental error. The results are compared with analogous data obtained for the five membered pyrrolidine ring of proline and with results for DNA itself.     

Diagnostic value of 9 am plasma adrenocorticotrophic hormone concentrations in Cushing's disease.

Morning plasma adrenocorticotrophic hormone (ACTH) concentrations were measured in 58 normal subjects and seven patients with pituitary-dependent Cushing''s syndrome (Cushing''s disease). Particular note was taken of the time of venepuncture. The range of values for the normal subjects irrespective of timing was 9-77 ng/l. The range between 9 0 am and 9 30 am was 9-24 ng/l. In the patients with Cushing''s disease the ACTH concentrations were in the range 39-109 ng/l. To distinguish patients with Cushing''s disease from normal subjects it is therefore important to define accurately the 9 am normal range, since these results show no overlap.     

Xylitol-mediated transient inhibition of ribitol utilization by Lactobacillus casei

The growth of Lactobacillus casei strain Cl-16 at the expense or ribitol was inhibited if the non-metabolizable substrate xylitol was included in the medium at concentrations of 6 mM or greater. At these concentrations, xylitol, did not competitively inhibit ribitol transport. The cessation of growth was caused by the intracellular accumulation of xylitol-5-phosphate, which occurred because growth on ribitol had gratuitously induced a functional xylitol-specific phosphotransferase system but not the enzymes necessary for the further metabolism of xylitol-5-phosphate. Eventually, the cells overcame the xylitol-mediated inhibition by repressing the synthesis of enzyme II of the xylitol phosphotransferase system so that xylitol-5-phosphate would no longer be accumulated within the cell.     

No Effect of Hyperketonemia on Local Cerebral Glucose Utilization in Conscious Rats

Abstract: Local cerebral glucose utilization was measured by the [¹⁴C]2-deoxy- d -glucose method in conscious control and hyperketonemic rats. Hyperketonemia was induced by 3 days of starvation or by infusion of 3- hydroxybutyrate in fed rats. These treatments produced combined blood ketone body concentrations (acetoacetate + 3-hydroxybutyrate) of from 1.2 to 2.4 mM. Neither treatment significantly affected glucose utilization in any of the 15 brain regions studied. These observations indicate that hyperketonemia in resting, conscious rats does not interfere with brain uptake and phosphorylation of glucose.     

Photosynthetic preparation and characterization of 13C-labeled carbohydrates in agmenellum quadruplicatum

The blue-green alga Agmenellum quadruplicatum (strain PR6) has been used to prepare photobiosynthetically ¹³C-labeled d-glucose, 2-O-(α-d-glucopyranosyl)-glyceric acid (glucosylglycerate), 2-hydroxy-1-(hydroxymethyl)ethyl α-d-gluco-pyranoside (glucosylglycerol), and α-d-glueopyranosyl β-d-fructofuranoside (sucrose). When grown to a cell density of 4.4 g.L^-1 (dry weight) under nitrate-nitrogen limiting growth conditions for 120 h, the algal cells contained 38% of the dry-cell weight as(1 → 4)-α-d-glucan (amylose). About 1% of the dry-cell weight was glucosylglycerol, glucosylglycerate, and sucrose. Glutamate was obtained, together with carbohydrates of low molecular weight, when the cells were extracted with chloroform-methanol; d-glucose was recovered from the extracted cells by acid hydrolysis of the starch. The algae were grown by using 20 mol% [¹³C] carbon dioxide for preparation of labeled carbohydrates and for cellular component identification by whole-cell n.m.r. spectroscopy.     

Determination of the mechanism and kinetic constants for hog kidney gamma-glutamyltransferase.

The initial-velocity kinetics of hog kidney gamma-glutamyltransferase were studied. Glutamate gamma-(4-nitroanilide) and its 3-carboxy derivative, glutamate gamma-(3-carboxy-4-nitroanilide), served as gamma-glutamyl donors, and glycylglycine as an acceptor. Reaction products were identified by paper chromatography and amino acid analysis. Inhibited Ping Pong mechanisms and a comprehensive initial- velocity expression were developed which account for the observed simultaneous gamma-glutamyl transfer and autotransfer, competitive inhibition by glycylglycine, and non-competitive inhibition by the carboxy donor. The validity of the proposed Ping Pong mechanisms are supported by enzyme-velocity data obtained with constant ratios of acceptor to donor concentrations. Kinetic constants were determined by a non-linear regression analysis. With glutamate gamma-(4-nitroanilide) as the donor, Michaelis constants for the donor, acceptor and donor-acting-as-acceptor are 1.87, 24.9, and 2.08 mM respectively. With glutamate gamma-(3-carboxy-4-nitroanilide) as the donor, these Michaelis constants are 1.63, 16.6, and 12.3 mM. Glyclyglycine competitive inhibition constants with the parent donor and its carboxy derivative are 275 and 205 mM respectively; the non-competitive inhibition constant of the carboxy donor is 34 mM.     

Aldolases of the lactic acid bacteria

Reciprocal qualitative and quantitative immunological experiments employing an anti-Pediococcus cerevisiae aldolase serum confirmed many of the interspecific relationships demonstrated previously among lactic acid bacteria with antisera prepared against the Streptococcus faecalis fructose diphosphate aldolase. The extent of immunological relatedness observed between the Lactobacillus and Pediococcus aldolases was markedly greater than that noted between Pediococcus and Streptococcus aldolases indicating that the pediococci share closer phylogenetic ties with the rod-shaped lactobacilli than with their spherical counterparts in the streptococci. In addition to confirming the existence of definitive, but distant, relationships between the lactic acid bacteria and certain gram positive nonsporeforming anaerobes, immunological cross-reactivity was also demonstrated between the pediococcal aldolases and those of Aerococcus viridans.This paper is dedicated with deepest appreciation to Prof. Roger Y. Stanier on the occasion of his 60th birthday in token of what his friendship and guidance have meant to me. — J. L.     

An abnormality of oestrogen feedback in amenorrhoea-galactorrhoea.

Fourteen patients with amenorrhoea and hyperprolactin-anemia but no evidence of pituitary tumours were each given an intramuscular injection of 1 mg oestradiol benzoate. Thirteen patients failed to release luteinizing hormone in response to the oestrogen. This hypothalamic abnormality may help to explain the menstrual disturbances in subjects with hyperprolactinaemia.     

13C Fourier transform nuclear magnetic resonance studies of fractionated Candida utilis membranes.

13C Fourier transform nuclear magnetic resonance has been used to study the lipid structure and dynamics of fractionated Candida utilis cell membranes. Measurements of the spin-lattice relaxation times indicate the existence of mobility gradients in the direction of increased mobility from the glycerol backbone toward the terminal methyl group of the fatty acid and toward the choline methyls. The temperature dependence of the relaxation times gives activation energies of approximately 4-6 kcal/mol for the rotations about various carbon-carbon bonds which determine the relaxation rates. In general, comparison with data which have been reported for artificial membrane systems indicates that the contributions of protein-lipid interactions to the T1 gradient are of negligible importance in the yeast membrane system. A dynamical model for the motion about bonds near unsaturated bonds which determined the relaxation of the unsaturated carbons is also proposed. Measurements of chemical shifts with temperature also exhibit a correlation with chain position. On the basis of these data a correlation of deltaE, the energy difference between gauche and anti conformations for gamma carboms, with chain position is inferred. In addition, an estimate of 1.2 kcal/mol can be obtained for deltaE for carbons near the end of the fatty acid chain. This value indicates that intermolecular interactions contribute substantially to deltaE since a value of approximately 0.5 kcal/mol can be ascribed to intramolecular interactions.