Volume 6, Issue 4
October 1984, pages 337-583
pp 337-348 October 1984
Alamethicin and related α-aminoisobutyric acid peptides form transmembrane channels across lipid bilayers. This article briefly reviews studies on the effect of alamethicin on lipid phase transitions in lipid bilayers and on mitochondrial oxidative phosphorylation. Fluorescence polarization studies, employing 1,6-diphenyl-1,3,5-hexatriene as a probe, suggest that alamethicin fluidizes lipid bilayers below the phase transition t-emperature, but has little effect above the gel-liquid crystal transition point. Alamethicin is shown to function as an uncoupler of oxidative phosphorylation in rat liver mitochondria. The influence of alamethicin on mitochondrial respiration is modulated by the phosphate ion concentration in the medium. Classical uncoupling activity is evident at low phosphate levels while inhibitory effects set in at higher phosphate concentrations. Time-dependent changes in respiration rates following peptide addition are rationalized in terms of alamethicin interactions with mitochondrial membrane components.
pp 349-355 October 1984
The unfolding-refolding of proteins is a cooperative process and, as judged by equilibrium properties, occurs in one step involving the native,N, and the unfoldedU, conformational states. Kinetic studies have shown that the denatured protein exists as a mixture of slow-(U)Sand fast-(U)Frefolding forms produced by proline peptidecis-trans isomerization. Proline residues inUFare in the same configuration as in the native protein while they are in non-native configuration inUS. For protein folding to occur quicklyUSmust be converted intoUF. The fact that the equilibrium and kinetic properties of are the same as those found for prolinecis-trans isomerization taken together with the absence of slow phase in the kinetics of refolding of a protein devoid of proline, support this view. However, the absence of a linear correlation between half-time of reactivation of denatured enzymes and their proline-contents, as well as the dissimilarities in the kinetic properties of in unfolding and refolding experiments are not consistent with the model. Conformational energy calculation and experimental results on refolding of proteins suggest that some proline residues are non-essential. They will not block protein folding even in wrong isomeric form. The native-like folded structure with incorrect proline isomers will serve as intermediate state(s) in which these prolines will more readily isomerize to the correct isomeric form. The picture becomes more complex when one considers the consequence ofcis-trans isomerism of non-proline residues on protein folding.
pp 357-377 October 1984
Although globular proteins are endowed with well defined three-dimensional structures, they exhibit substantial mobility within the framework of the given three-dimensional structure. The different types of mobility found in proteins by and large correspond to the different levels of organisational hierarchy in protein architecture. They are of considerable structural and functional significance, and can be broadly classified into (a) thermal and conformational fluctuations, (b) segmental mobility, (c) interdomain mobility and (d) intersubunit mobility. Protein crystallographic studies has provided a wealth of information on all of them. The temperature factors derived from X-ray diffraction studies provide a measure of atomic displacements caused by thermal and conformational fluctuations. The variation of displacement along the polypeptide chain have provided functionally significant information on the flexibility of different regions of the molecule in proteins such as myoglobin, lysozyme and prealbumin. Segmental mobility often involves the movement of a region or a segment of a molecule with respect to the rest, as in the transition between the apo and the holo structures of lactate dehydrogenase. It may also involve rigidification of a disordered region of the molecule as in the activation of the zymogens of serine proteases. Transitions between the apo and the holo structures of alcohol dehydrogenase, and between the free and the sugar bound forms of hexokinase, are good examples of interdomain mobility caused by hinge-bending. The capability of different domains to move semi-independently contributes greatly to the versatility of immunoglobulin molecules. Interdomain mobility in citrate synthase appears to be more complex and its study has led to an alternative description of domain closure. The classical and the most thoroughly studied case of intersubunit mobility is that in haemoglobin. The stereochemical mechanism of the action of this allosteric protein clearly brings out the functional subtilities that could be achieved through intersubunit movements. In addition to ligand binding and activation, environmental changes also often cause structural transformations. The reversible transformation between 2 Zn insulin and 4 Zn insulin is caused by changes in the ionic strength of the medium. Adenylate Kinase provides a good example for functionally significant reversible conformational transitions induced by variation in pH. Available evidences indicate that reversible structural transformations in proteins could also be caused by changes in the aqueous environment, including those in the amount of water surrounding protein molecules.
pp 379-401 October 1984
Bacterial citrate lyase, the key enzyme in fermentation of citrate, has interesting structural features. The enzyme is a complex assembled from three non-identical subunits, two having distinct enzymatic activities and one functioning as an acyl-carrier protein. Bacterial citrate lyase,si-citrate synthase and ATP-citrate lyase have similar stereospecificities and show cofactor cross-reactions. On account of these common features, the citrate enzymes are promising markers in the study of evolutionary biology. The occurrence, function, regulation and structure of bacterial citrate lyase are reviewed in this article.
pp 403-418 October 1984
The aspartate pathway of amino acid biosynthesis in bacteria serves as paradigm for the evolution of patterns of enzyme regulation in response to specific physiological requirements. InBacillus species, the first step in the pathway is catalyzed by multiple forms of aspartokinase, which differ in their structure and feedback regulation. One form of aspartokinase (V-type) functions primarily during cell growth, another form (S-type) during sporulation. The V-type aspartokinase fromBacillus subtilis andBacillus polymyxa is discussed in some detail on account of its complex pattern of regulation by the pathway endproducts lysine and threonine and its unusual subunit structure. The enzyme is composed of two dissimilar subunits, the smaller of which corresponds to the carboxyl-terminal domain of the larger subunit. The coding sequence for the subunits ofBacillus subtilis aspartokinase has recently been cloned inEscherichia coli. The study of its structure and mode of expression has revealed that the two aspartokinase subunits are encoded by in-phase overlapping genes. These unusual features of aspartokinase suggest that important aspects of the regulation of the aspartate pathway are yet to be discovered.
pp 419-430 October 1984
Since the recognition of the ‘translocation’ phenomenon during protein synthesis several theories have been proposed, without much success, to explain the translocation of peptidyl tRNA from the aminoacyl site to the peptidyl site. The involvement of L7/L12 proteins and therefore the L7/L12 stalk region of 50S ribosomes in the translocation process has been widely accepted. The mobility of the stalk region, as recognised by many workers, must be of physiological significance. It has recently been shown in this laboratory that 50S ribosomes derived from tight and loose couple 70S ribosomes differ markedly in quite a few physical and biological properties and it appears that these differences are due to the different conformations of 23S RNAs. It has also been possible to interconvert tight and loose couple 50S ribosomes with the help of the agents, elongation factor -G, GTP (and its analogues) which are responsible for translocation. Thus loose couple 70S ribosomes so long thought to be inactive ribosomes are actually products of translocation. Further, the conformational change of 23S RNA appears to be responsible for the interconversion of tight and loose couple 50S ribosomes and thus the process of translocation. A model has been proposed for translocation on the basis of the direct experimental evidences obtained in this laboratory.
pp 431-457 October 1984
Microtubules are ubiquitous cellular structures found in eukaryotic organisms and responsible for a variety of functions. These functions include mitosis, motility, cytoskeletal architecture, intracellular transport and secretion. The major structural component of microtubules is tubulin, a dimeric protein molecule consisting of two similar but nonidentical subunits (α and β) each of about molecular weight 55,000. With the introduction of radioactive colchicine for the first time it has been reported that colchicine binds specifically to tubulin. At this point microtubule research stepped up to a new era linking microtubules with other spindle poisons which are structurally diverse as well as binding at different sites on to the tubulin heterodimer. These antimicrotubular agents have already provided valuable information regarding microtubule-mediated cellular functions and its association and dissociation phenomena. Tubulins appear to be conserved proteins based onin vitro copolymerization and comigration on polyacrylamide gel electrophoretic properties. Further, amino acid sequences of both α and β subunits from a variety of sources also appear to be mostly conserved. The evolutionary conservation of tubulin genes is highly reflected at the nucleic acid level as well. The estimation of the number of genes for tubulin and their organization in a variety of organisms have opened up a new dimension to microtubule and tubulin research. The multigene family for tubulins comprising also pseudogenes is suggestive that more than one gene for each α and β tubulin is functional in the cell. Therefore, it has been speculated that different tubulin gene products contribute to functionally different microtubules at specific stages in cell cycle and cell growth. Heterogeneity in both α andβ tubulins has already been established during different stages of development of the cell. Obviously, it reflects that tubulin genes are highly regulated and this regulation might be at the transcriptional and/or translational level. Whatever is the actual control mechanism it appears that cells can detect an enhanced pool of depolymerized subunits and a rapid and specific control in tubulin gene expression at the transcriptional and/or post transcriptional level does occur.
pp 459-474 October 1984
While the dietary importance of proteins, essential fatty acids, vitamins and trace elements has been well recognised, the role of shadow nutrients, a class of metabolites, which are biosynthesized in the body and serve vital functions, such as lipoic acid, choline, inositol, taurine and carnitine, has not been adequately appreciated. There are reasons to believe that during infancy and in ageing, biosynthesis of these metabolites may be limited. The objective of this review is to highlight the essentiality of these nutrients and the need for their supplementation in the diets of infants and in elderly people. Provision of shadow nutrients where the necessary biosynthetic machinery might not have developed to full stature or might have slowed down, is a new concept in nutrition which needs attention.
pp 475-489 October 1984
Differentiation into dormant cysts and vegetative trophozoites is an inherent character intimately associated with the life cycle and infectivity of pathogenic amoebae. In the case of human intestinal amoebiasis encystation and excystation are of immediate relevance to the process of transmission of the disease from healthy carriers to susceptible individuals. Using a pathogenic free living amoebaAcanthamoeba culbertsoni as a model, considerable progress has been achieved in understanding the mechanism and control of the process of differentiation. The turnover of the regulatory molecule cyclic 3: ’5′ adenosine monophosphate is responsible for triggering the process of encystation. Amoebae bind effector molecules such as biogenic amines to a membrane localized receptor which itself resembles the β-adrenergic receptor of mammalian organisms. The activation of adenylate cyclase or inhibition of cyclic AMP phosphodiesterase maintain the dynamic intracellular cyclic AMP. The cytosol fraction of amoebae has a cyclic AMP binding protein. During encystation, enzymes needed for synthesis of cellulose and glycoproteins are induced. Control is exercised at transcriptional level and the process is subject to catabolic repression.
Excystation of mature amoebic cysts is mediated by glutamic acid and certain other amino acids by an as yet unelucidated mechanism. During excystation there is dormancy break, induction of deploymerizing enzymesviz. two proteases, a cellulase and a chitinase. The empty cysts or cyst walls are digested by these enzymes and their break down products are used for cellular growth. By invoking a flip-flop mechanism of repression and derepression some plausible explanation can be offered for the cascade of biochemical events that sets in when amoeba is ‘turned on’ to encystation or excystation.
pp 491-502 October 1984
Liposomes, the artificial phospholipid vesicles, have the capacity of entrapping water soluble substances in their aqueous compartments. Of the many possible potentials of liposomes their application in immunology is most significant. Recent studies have shown an adjuvant and a carrier effect of liposomes to a number of antigens. Liposomes used in these studies are generally multilamellar vesicles with the antigen encapsulated in the aqueous phase. Some antigens may also be associated with the lipid lamellae covalently or noncovalently. The adjuvant property of liposomes is greatly affected by the surface charge of the vesicle as well as the site of association of the antigen. The other factors which may have a role in immunopotentiation by liposomes are the size and structure of the vesicles, the lipid composition, route of administration and their surface sugars. In addition, liposomes may function as carriers to haptens and other antigens. In association with liposomes the nature of the immune response may be modulated. For a further enhancement of the adjuvant activity of liposomes use has been made of immunomodulators.
pp 503-524 October 1984
This article considers ways in which various genes are regulated and the approaches that have been used to isolate the gene regulatory proteins involved. Isolation of regulatory proteins uses standard techniques for protein purification. The main problem in the isolation is not these techniques but rather assay procedures that permit detection of the regulatory protein during the course of isolation. For this purpose assays that are most useful are those that combine selectivity with sensitivity. In prokaryotes there is no better assay than that provided by the coupled transcription translation system. Plasmids containing regulatory genes can frequently be used as abundant sources of regulatory proteins and sometimes even permit the direct detection of the regulatory protein. In eukaryotes the situation is far more difficult because the isolation of regulatory genes is much more difficult. It seems likely that selective transcription and selective DNA binding will provide the most useful assays.
pp 525-533 October 1984
Certain species of bacteria can become competent to take up high molecular weight DNA from the surrounding medium. DNA homologous to resident chromosomal DNA is transported, processed and recombined with the resident DNA. There are some variations in steps leading to transformation between Gram-positive bacteria likebiplococcus pneumoniae and Gram-negative bacteria represented byHaemophilus influenzae but the integration is by single-strand displacement in both cases. Plasmid (RSF0885) transformation is low inHaemophilus influenzae but this is increased significantly if (homologous) chromosomal DNA is spliced to plasmid DNA. InHaemophilus influenzae, rec1 function is required for peak transformation with chimeric plasmids. Chimeric plasmid fixed presumably extrachromosomally undergoes frequent recombination between homologous segments contained in resident chromosome and the plasmid.
pp 535-542 October 1984
The decade of the 70’s was remarkable for the insights that rapidly accumulated to provide us with an understanding of one of the fundamental processes of animal cell metabolism, namely, how mammalian cells ingest a host of extracellular substances to satisfy their various metabolic needs. It has long been appreciated that the surfaces of mammalian cells are in a continual state of flux. Surface membranes often fold inward and pinch of in a vesicular form trapping some of the contents of the extracellular material which are thus transported into the cell. This process is called endocytosis (reviewed in Silversteinet al., 1977). When extracellular fluids are taken up in this manner, the process is called fluid-phase endocytosis or pinocytosis. When solids are ingested, the process is called phagocytosis. Although quantitatively important over the long run, these modes of uptake are slow, non-specific and dependent on the concentration of the substance in the extracellular medium. In recent years it has been recognized that animal cells have developed a specialized form of this vesicular transport system to selectively retrieve and assimilate macromolecules from the extracellular milieu with high efficiency. This process is called receptor-mediated endocytosis. In this review an attempt is made to collate and correlate the evidence establishing receptor-mediated endocytosis as a dynamic process that routes cell surface receptors and ligands through multiple intracellular compartments to their ultimate destination.
pp 543-567 October 1984
There are several methods to introduce purified DNA into mammalian cells. These include microinjection into the nuclei of the recipient cells and complexing the DNA with facilitating agents such as calcium-phosphate. After it enters the nucleus, the DNA is expressed in a large proportion of the cells. This expression is dependent upon cis-acting sequences that are recognized by the mammalian transcriptional and translational machinery. In a smaller proportion of cells, the exogenous DNA becomes covalently integrated into the host cell DNA at random sites. Non-selectable genes can be introduced into mammalian cells by ligating them to a selectable marker or mixing the DNA with carrier DNA containing a selectable marker. The DNA that is introduced into mammalian cells can be rescued for examination and analysis. These gene transfer methods have already proven to be useful in identification of sequences which are necessary for normal gene expression as well as gene regulation. In addition a number of genes have been isolated using gene transfer methods. DNA mediated gene transfer holds much promise to target genes to specific sites in the chromosomes, to understand mechanisms of mammalian development and cell differentiation and is expected to provide a method to produce important and novel gene products that may be used for diagnostic and therapeutic purposes.
pp 569-583 October 1984
The effect of viruses on plasma membrane function has been studied in two types of situation: (i) during the toxin-like action of paramyxoviruses when fusing with susceptible cells, and (ii) during an infectious cycle initiated by different viruses in various cell types.
The nature of the permeability changes induced during the toxin-like action of viruses, and its modulation by extra-cellular Ca2+, are described: membrane potential collapses, intracellular ions and metabolites leak out of, and extracellular ions leak into cells, but lysis does not take place. The biological significance of such changes, and their relation to changes induced by other pore-forming agents, are discussed.
Changes in membrane permeability such as those mentioned above have not been detected during infection of cultured cells by paramyxo (Sendai, measles, mumps), orthomyxo (influenza), rhabdo (vesicular stomatitis), toga (Semliki Forest) or herpes viruses. On the contrary, sugar uptake is increased when BHK cells are infected with vesicular stomatitis virus, semliki forest virus or herpes virus. Cultured neurones infected with herpes simplex virus show changes in electrical activity. The pathophysiological significance of these alterations in membrane function, which occur in viable cells, is discussed.
It is concluded that clinical symptoms may result from cell damage caused by virally induced alterations of plasma membrane function in otherwise intact cells.