No abstract

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.Cyclic AMP (cAMP)-dependent protein kinase (cAMPdPKase; ATP:protein phosphotransferase, EC 2.7.1.37) (1) can be regarded as a major intracellular sensor ofchanges in cAMP levels, which take place in response to extracellular hormonal stimuli. As such, it is in charge of implementing the instructions given by a hormone to its target cell. This enzyme has been found in quite a few mammalian tissues (2) and is composed of two types of subunits, one being catalytically active (C) and the other having a regulatory function (R) (3, 4). These two subunits are assembled together to yield the inactive form of the enzyme, R2C2, which is now believed to be activated by cAMP according to the following equation (5-7):R2C2 + 4cAMP = R2(cAMP)4C2± R2(cAMP)4 + 2C. One of the intriguing questions regarding the physiological function of cAMPdPKase arises from the fact that this enzyme can phosphorylate various proteins, at least in vitro and probably even in intact cells (8). Therefore, it becomes imperative to elucidate the mechanism(s) by which the kinase activity may be channeled in vivo to bring about a specific phosphorylation at a given time and a given locus within the cell in response to a distinct metabolic signal. Some reports have suggested that different hormones that function through cAMP may trigger different chains of metabolic events within the same cell (9). This raises the possibility that there may be additional regulatory devices for modulating the cellular response to the hormonal stimulus.cAMPdPKase activity in brush-border membranes (from the rat small intestine) vanishes within a few minutes upon addition of cAMP (10). The inactivation was shown to be ...

No abstract

Intact erythrocytes become immediately crenated upon addition of 2,4-dinitrophenol (DNP) or pyrenebutyric acid (PBA) . However, when cells are incubated at 37°C in the presence of the crenating agents with glucose, they gradually (4-8 h) recover the normal biconcave disc form . The recovery process does not reflect a gradual inactivation of DNP or PBA since fresh cells are equally crenated by the supernatant from the recovered cells. Further, after recovery and removal of the crenating agents, cells are found to be desensitized to the readdition of DNP as well as to the addition of PBA, but they are more sensitive to cupping by chlorpromazine . This alteration in the cell \membrane responsiveness was reversible upon further incubation in the absence of DNP. Recovery s dependent upon cellular metabolic state since an energy source is needed and incubation with uanosine but not adenosine will accelerate conversion to the disc shape. It is suggested that the conversion of cells from crenated to disc shape in the presence of the crenators, represents an alteration or rearrangement of membrane components rather than a redistribution of the crenators within the membrane . This shape recovery process may be important for erythrocyte shape preservation as well as shape control in other cells .

On the basis of recent studies a model of erythrocyte shape control is presented. This model is based on the assumption that the erythrocyte shape is normally determined by relative surface tensions of the two membrane bilayer surfaces. Our studies have indicated that the erythrocyte shape is preserved and can be restored upon perturbation by an active process. Consequently, it is proposed that in the "bilayer sensor" model the cells "sense" changes in the relative surface tensions, activating a process that balances the tensions so as to create a biconcave disc shape (reaching a new steady state which is observed as long as the perturbant is present). Experimental evidence has indicated that there is a mechanism to sense shape perturbations and two mechanisms to change shape through either crenation or cupping.