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Biomedical subjects

S Shaltiel

Publications and source records attributed to S Shaltiel.

At least 55 records · Page 3Linked to original sources
PubMed1984

Hydrophobic chromatography of cells: adsorption and resolution on homologous series of alkylagaroses.

Implantation of hydrocarbon chains on beaded agarose results in column materials capable of adsorbing erythrocytes or lymphocytes from various sources. In a homologous series of such columns, identical in all structural respects (ligand density, charge density and ultrastructure) except for the length of their hydrocarbon chains, the capacity to adsorb these cells generally increases with increase in the number of carbon atoms per chain. The cells can be desorbed from the columns by repeated gentle pipetting in the presence of bovine serum albumin (for erythrocytes) or foetal calf serum (for lymphocytes). Under the conditions used for adsorption and desorption there is neither physical entrapment of the cells in the column nor apparent damage to their integrity, as indicated by the facts that (a) 95-98% of the cells applied on the column can be recovered; (b) erythrocytes eluted from the column and those yet to be applied are morphologically indistinguishable, exhibit an identical osmotic fragility profile and, (after desorption) retain the same adsorption profile for the columns; and (c) over 95% of the mouse spleen lymphocytes, eluted from the columns, continue to exclude the dye Trypan blue, suggesting that they preserve their viability. Cells from different sources exhibit different adsorption profiles on homologous series of alkylagaroses, allowing the detection of differences in the surface of these cells and their resolution by these columns. Exploratory experiments with artificial cell mixtures are described, illustrating the possibility of enriching the mixture with one of the cell types by preferential adsorption or exclusion on an appropriate alkylagarose. The best resolution was obtained with a mixture of erythrocytes and spleen lymphocytes (both from DBA/1 mice). After appropriate "tailoring" of the column material used (an ethylagarose) it was possible to apply a 1:1 mixture of these cells and to obtain (after preferential adsorption) essentially pure erythrocytes (98% of the excluded cells).(ABSTRACT TRUNCATED AT 400 WORDS)

Adsorption↗
PubMed1983

Viable mouse thymocytes as a model system for studying the onset of hormone-induced cellular refractoriness.

Mouse thymocytes are characterized as a model cellular system for studying the onset of hormone-induced cellular refractoriness (desensitization). This system has the following combination of useful features. (a) The cells can be isolated without the use of digestive enzymes, avoiding possible damage to surface receptors or to other exposed membranal constituents. (b) They can be kept viable for several hours, a period during which both stimulation and desensitization get well under way. (c) They can be stimulated by a variety of hormones which function via cAMP (beta-agonists, prostaglandin E1 and specific thymic humoral factors). (d) Their desensitization is receptor-specific. (e) They can be readily ruptured under mild conditions so as to allow a physiologically relevant biochemical analysis of hormonal stimulation and desensitization. (f) The hormonal response of these cells can be monitored simultaneously by the activation of adenylate cyclase, by the intracellular level of cAMP, and by the activation of cAMP-dependent protein kinase (which functions as a metabolic sensor for cAMP). In this cellular system, desensitization does not involve processes such as the efflux of cAMP, the activation of cAMP-phosphodiesterase or the synthesis of a protein mediator. On the other hand, desensitization can be accounted for by a hormone-triggered inactivation of the adenylate cyclase system. The immediate desensitization of thymocytes is reversible and occurs without apparent loss of functional receptors. Continuous presence of hormone is shown to be required not only for triggering the chain of events which leads to the readily reversible desensitization, but also for the process which transfers the cells to the subsequent, 'locked' desensitized state.

Adenylyl Cyclases↗
PubMed1982

Exposure of thymocytes to a low temperature (4 degrees C) inhibits the onset of their hormone-induced cellular refractoriness.

The hormonal response of viable mouse thymocytes is radically dependent of their ambient temperature. While at 37 degrees C the cells respond to isoproterenol by an abrupt rise (within 30 s) followed by a exponential decline in the level of intracellular cAMP, at 4 degrees C the level of cAMP remains high, i.e. there is an inhibition of the hormone-induced refractory state. These distinctly different patterns of response are reflected also in both the state of activation of cAMP-dependent protein kinase and the activity of adenylate cyclase. The inhibition of cellular refractoriness in the cold is shown to be fully reversible, lasting only as long as the hormone is present in the extracellular medium. Washing out the hormone or displacing it by a specific antagonist (propranolol) results in a decline of cAMP, of the activity ratio of the kinase, and of the activity of the adenylate cyclase back to basal values. Evidence is presented to show that at 4 degrees C there is no significant hormone-dependent decreases in cAMP degradation or efflux. On the other hand, the activity of adenylate cyclase remains persistently high, through neither the hormone-binding site of the receptor nor the active site of the catalytic subunit of the cyclase seem to be impaired. The different response pattern observed at 4 degrees C appears, therefore, to be associated with the transfer and the signal between these two sites and probably with the G/F protein (s). The possibility to dissect in a selective and reversible manner the process of hormonal stimulation (coupling) from the process of desensitization, which, under normal physiological conditions constitute consecutive and inseparable chain of events, leads us to a propose that the signal transfer which enables activation of adenylate cyclase is, somewhere along its way, distinct from the signal transfer which brings about the onset of the refractory state, and to conclude that these two processes are partially autonomous and regulated by either two different proteins or two different sites on the same protein. The postulated proteins (or sites) should, therefore, differ in their sensitivity to temperature changes, a difference which may be most useful in the identification and isolation of the molecular species involved and in the study of their properties and their mechanism of action.

Adenylyl Cyclases↗
PubMed1981

Degradative inactivation of cyclic AMP-dependent protein kinase by a membranal proteinase is restricted to the free catalytic subunit in its native conformation.

A membranal proteinase from brush-border epithelial cells of the rat small intestine was shown to bring about a restricted and limited degradation of the free catalytic subunit (C) of cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) with concomitant inactivation of the kinase. This membranal proteinase exhibits a remarkable specificity. (i) It degrades C in its native conformation, but not after it has been heat-denatured. (ii) The degradation of C (Mr 40,000) does not proceed further, once a distinct clipped product (Mr 34,000) is formed. (iii) The undissociated ("stored") form of the enzyme (R2C2) is not attacked by the membranal proteinase, preserving both its potential catalytic activity and its molecular integrity. Only upon addition of cyclic AMP to release free C does the proteinase attack it. (iv) The membranal proteinase does not degrade the regulatory subunit (R), released by cyclic AMP from R2C2, although R is quite susceptible to degradation by other proteolytic enzymes. None of these features of the membranal proteinase could be reproduced with trypsin, chymotrypsin, clostripain, or papain. The specific, restricted, and limited action of this membranal enzyme raises the possibility that it may have a distinct physiological assignment associated with the bioregulation of cyclic AMP-dependent protein kinase.

Animals↗
PubMed1980

Inhibiting the onset of hormone-induced desentiziation of viable thymocytes by N alpha-tosyl-L-lysine chloromethyl ketone.

N alpha-Toysl-L-lysine chloromethyl ketone (Tos-LysCH2Cl) was found to inhibit irreversibly the onset of the hormone-induced refractory state in intact thymocytes. When thymocytes (approximately 2 X 10(7) cells per ml) are treated with Tos-LysCH2Cl(10(-4) M, for 90 min at pH 7 and 37 degrees C) the cells retain their viability, including a full capacity to recognize and respond to hormonal stimuli, yet they selectively lose their ability to become desensitized to persistent triggering by a hormone, as reflected in the state of activation of intracellular cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). Whereas upon hormonal stimulation of untreated cells the immediate rise in the state of activation of this enzyme (up to an activity ratio of > 0.85) is followed by an exponential decline to basal values within approximately 60 min, in TosLysCH2Cl-treated cells the hormone-triggered elevation in the state of activation of the enzyme is maintained for > 60 min. Evidence is presented to suggest that in thymocytes TosLysCH2Cl inhibits the regulatory process that normally uncouples the adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] system without interfering with previous or subsequent molecular events connected with the transfer of hormonal signals across the cell membrane. This technique allows, therefore, the preparation of viable thymocytes with a limited and distinct regulatory defect introduced by chemical (covalent) means. As such, it is most useful for studies aimed at the elucidation of the mechanism of cell desensitization and for further characterization and localization of key components responsible for cellular refractoriness.

Amino Acid Chloromethyl Ketones↗
PubMed1979

Affinity labeling of the catalytic subunit of cyclic AMP-dependent protein kinase by N alpha-tosyl-L-lysine chloromethyl ketone.

The catalytic subunit of cyclic AMP-dependent protein kinase (from rabbit skeletal muscle; ATP:protein phosphotransferase, EC 2.7.1.37) was found to be irreversibly inactivated by chloromethyl ketone derivatives of lysine and phenylalanine, chemical reagents originally designed for labeling the active sites of the proteolytic enzymes trypsin and chymotrypsin. This inactivation was shown to occur at pH 7.5 and 22 degrees C, conditions under which chemically related alkylating reagents such as chloroacetamide and chloroacetic acid (which do not possess the amino acid side chain) fail to inactivate the enzyme. In the case of the chloromethyl ketone derivative of N alpha-tosyl-L-lysine, the enzyme could be protected by its nucleotide substrate (MgATP), by one of its protein substrates (histone H2b), and by its regulatory subunit which, upon binding, shields the active site of the catalytic subunit. Differential labeling experiments, together with kinetic studies of the rates of modification of the sulfhydryl groups in the enzyme before and after inactivation with the chloromethyl ketone, suggest that the loss of activity is associated with one (kinetically characterized) sulfhydryl group present either at the active site of the enzyme or at a site intimately associated with it. The general implications of these results regarding the interpretation of affinity labeling experiments carried out in complex mixtures of proteins or under in vivo conditions are discussed.

Affinity Labels↗
PubMed1978

Mapping the ATP-binding site in the catalytic subunit of adenosine-3':5'-monophosphate-dependent protein kinase. Spatial relationship with the ATP site of the undissociated enzyme.

A set of 24 ATP analogs modified at various positions of the ATP molecule was used for mapping the ATP-binding site in the free catalytic subunit (C) of cAMP-dependent protein kinase (type I). Ki values for these analogs (of which 23 were shown to be competitive with ATP) were measured and compared with Ki values previously obtained for the same set of analogs upon binding to the undissociated form of the enzyme (R2C2). It was found that modifications at the adenine part of ATP bring about a considerable reduction in affinity between C and the resulting analog. The other parts of the ATP molecule play a less important, though definite, role in the binding of this nucleotide to C. By measuring the effect of each given modification in ATP on its binding to C, and comparing the effect of this modification on the binding of the same analog to R2C2, it was possible to obtain 'specificity profiles' for both forms of the kinase. Using such profiles it is shown that the adenine-binding subsite in C may well coincide with the adenine-binding subsite in R2C2. Two plausible models describing the spatial relationship between the ATP sites in C and R2C2 are proposed.

Adenosine Triphosphate↗