Multiple conformations of amino acid residues in ribonuclease A

The highly refined 1.26 A structure (R = 0.15) of phosphate-free bovine pancreatic ribonuclease A was modeled with 13 residues having discrete multiple conformations of side chains. These residues are widely distributed over the protein surface, but only one of them, Lys 61, is involved in crystal packing interactions. The discrete conformers have no unusual torsion angles, and their interactions with the solvent and with other atoms of the protein are similar to those residues modeled with a single conformation. For three of the residues--Val 43, Asp 83, and Arg 85--two correlated conformations are found. The observed multiple conformations on the protein surfaces will be of significance in analyzing structure-function relationships and in performing protein engineering.     

Dye permeability at phase transitions in single and binary component phospholipid bilayers

By encapsulating a pH-sensitive dye, phenol red, in multilamellar liposomes of DMPC, DPPC and DMPC/DPPC mixtures, the permeability of these phospholipid bilayers to dye as a function of temperature has been studied. For both DMPC and DPPC liposomes, dye release begins well below the main gel-to-liquid-crystalline phase transition (24°C and 42°C, respectively) at temperatures corresponding to the onset of the pretransition (about 14°C and 36°C, respectively) with DPPC liposomes exhibiting a permeability anomaly at the main phase transition (42°C). The perturbation occurring in the bilayer structure that allows the release of encapsulated phenol red (approx. 5 Å diameter) is not sufficient to permit the release of encapsulated haemoglobin (approx. 20 Å diameter, negatively charged). In liposomes composed of a range of DMPC/DPPC mixtures, dye release commences at the onset of the pretransition range (determined by optical absorbance measurements) and increases with increasing temperature until the first appearance of liquid crystalline phase after which no further dye release occurs. Interestingly, the dye retaining properties of DMPC and DPPC liposomes well below their respective pretransition temperature regions are very different: DMPC liposomes release much encapsulated dye at incubation temperatures of 5°C whilst DPPC liposomes do not.     

Isolation and partial characterization of the basal cell membrane of human placental trophoblast

The function of the syncytiotrophoblast in maternal-fetal exchange is related to the properties of its microvillous (maternal-facing) and basal (fetal-facing) plasma membranes. We have previously reported the properties of the microvillous membrane (Smith, C.H., Nelson, D.M., King, B.F., Donohue, T.M., Ruzycki, S.M. and Kelley, L.K. (1977) Am. J. Obstet. Gynecol. 128, 190–196), and now describe the purification and partial characterization of the basal plasma membrane. Sonication and incubation with EDTA were used to isolate selectively the basal cell membrane. These steps were followed by a more conventional purification by centrifugation. The trophoblast was disrupted and its microvillous membrane and cytoplasmic contents were removed by sonication. The exposed basal cell membrane was selectively released from the underlying basal lamina by sonication in the presence of EDTA and further purified by discontinuous Ficoll gradient centrifugation. The material at the 4–10% Ficoll interface consisted of smooth membrane vesicles with internal microfilaments. It was 45-fold enriched in dihydroalprenolol binding activity and 11-fold enriched in ouabain binding activity. Other enzymatic analyses, including alkaline phosphatase, cytochrome-c oxidase, cytochrome-c reductase and galactosyl transferase indicated low contamination by other organelles. This procedure yields a preparation of relatively high purity which should be suitable for investigation of transport and other functions of the basal surface membrane of trophoblast. In principle, the purification procedures used may be applicable to other transporting epithelia.     

Effect of Elevated Temperature on Extended Enzyme Synthesis Induced by Bacteriophage T4 Amber Mutants Unable to Synthesize Deoxyribonucleic Acid

The extended synthesis of early enzymes by the deoxyribonucleic acid-negative amber mutants of bacteriophage T4 after infection of the nonpermissive host Escherichia coli B was prevented by incubating the infected cells at 44 C. This effect did not occur if the incubation temperature was 43 C or less or if the cells were grown and infected in broth rather than minimal medium (C medium). Once early enzyme synthesis had ceased at 44 C, lowering the incubation temperature to 37 C did not occasion resumption of synthesis. Experiments with chloramphenicol at 44 C indicated that increased degradation of early enzymes is an unlikely explanation for the effect. Examination of pulse-labeled ribonucleic acid and polysomes made at 37 and 44 C in infected cells revealed some differences, but at present there is no obvious way in which these differences may be related to the effect on enzyme formation. There was no discernible difference between the ribosomal ribonucleic acid and ribosomes at the two temperatures, nor was there a difference in the cell-free amino acid-incorporating systems isolated from cells infected at the two temperatures as judged by polyuridylic stimulation of phenylalanine incorporation. Incubation of cells infected with T4amN82 at 44 C with protein synthesis blocked by 5-methyltryptophan for 15 min did not prevent the typical pattern of enzyme synthesis at 44 C when the block was reversed by excess l-tryptophan. The relation of this and other observations relative to the effect at 44 C on the synthesis of early enzymes is discussed.     

Regulation of Newly Evolved Enzymes. I. Selection of a Novel Lactase Regulated by Lactose in ESCHERICHIA COLI

Thirty-four lactose-utilizing strains of E. coli were selected from a lac Z deletion strain. In 31 of these, the synthesis of the newly evolved lactase is regulated by lactose. The lactase activity in all the strains is indistinguishable from the ebg(+) activity identified by Campbell, Lengyel and Langridge (1973).     

The effects of potassium, 5-hydroxytryptamine, adrenocorticotrophin and angiotensin II on the concentration of adenosine 3′:5′-cyclic monophosphate in suspensions of dispersed rat adrenal zona glomerulosa and zona fasciculata cells

Dispersed rat adrenal cells prepared from both the capsule and the decapsulated gland were used to investigate the effects on cyclic AMP accumulation of known stimuli of steroidogenesis [ACTH (adrenocorticotrophin), angiotensin II, K(+) ions and 5-hydroxytryptamine]. Since glomerulosa-cell preparations from capsular strippings are normally contaminated with a proportion of fasciculata cells, cells purified by fractionation on a bovine serum albumin gradient were also used. The results showed that: (1) ACTH and angiotensin II stimulated cyclic AMP accumulation in both fractionated and unfractionated zona fasciculata cells; (2) 5-hydroxytryptamine and an increased extracellular K(+) concentration (from 3.6 to 8.4mm) had no effect on cyclic AMP concentrations in fasciculata cell preparations; (3) the addition of ACTH, angiotensin II, 5-hydroxytryptamine or K(+) to the incubation medium resulted in increased cyclic AMP concentrations in unpurified zona glomerulosa cell preparations; (4) fractionation and hence the virtual elimination of fasciculata contamination, did not affect the response to 5-hydroxytryptamine and increased K(+) concentration. However, the responses to ACTH and angiotensin II were markedly lowered but not abolished. These results strongly suggest a link between cyclic AMP production and steroidogenesis in the zone of the adrenal gland that specifically secretes aldosterone. All four agents used stimulated both steroid output and cyclic AMP accumulation. However, at certain doses of 5-hydroxytryptamine, K(+) and angiotensin II the significant increases in corticosterone output were not accompanied by measurable increases in cyclic AMP accumulation.     

Analysis of the code relating sequence to conformation in globular proteins. Theory and application of expected information

1. An information theory analysis of the folding of a globular protein is proposed. 2. The folding is seen as a transfer of information between two messages, the primary sequence and the biologically active conformation. 3. It is shown how the information transferred was estimated by inspection of proteins of known primary sequence and conformation. 4. In this estimation, concerted use of subjective (Bayesian) probabilities leads to a more robust approach which can be employed whether the number of proteins of known sequence and conformation is large or small. 5. Further, it is demonstrated that the problem then becomes a very simple algebraic formulation for information estimates. 6. Finally, it is shown how this process of information theory analysis can be reversed to predict the conformation of a protein by using its primary sequence and the above information estimates obtained from other proteins. 7. The present paper provides the theoretical basis for the derivation and application of a stereochemical alphabet (Robson & Pain, 1974a,c), and for an investigation of the effects of residues on the conformations of their neighbours (Robson & Pain, 1974b).     

Selective Utilization of Pyrimidine Deoxyribonucleosides for Deoxyribonucleic Acid Synthesis in Pneumococcus

When pyrimidine deoxyribonucleosides are supplied to growing cultures of Diplococcus pneumoniae, they are selectively used for incorporation into deoxyribonucleic acid (DNA). Differently labeled molecules of deoxyuridine, thymidine, and deoxycytidine were used to study the precursor pathways of this organism. Each of these preformed pyrimidine deoxynucleosides is incorporated intact (i.e., without cleavage of the glycosidic bond) and is predominantly recoverable as DNA thymidine. During the utilization of deoxycytidine and deoxyuridine by pneumococci, large proportions of the available precursor are converted to free thymidine, which is secreted back into the growth medium. The biochemical pathways for selective incorporation into DNA and the regulation of concentrations of intracellular thymidine compounds by excretion of free thymidine are discussed.