Collagen-tailed asymmetric acetylcholinesterase (AChE) forms are believed to be anchored to the synaptic basal lamina via electrostatic interactions involving proteoglycans. However, it was recently found that in avian and rat muscles, high ionic strength or polyanionic buffers could not detach AChE from cell-surface clusters and that these buffers solubilized intracellular non-junctional asymmetric AChE rather than synaptic forms of the enzyme. In the present study, asymmetric AChE forms were specifically solubilized by ionic buffers from synaptic basal lamina-enriched fractions, largely devoid of intracellular material, obtained from the electric organ of Torpedo californica and the end plate regions of rat diaphragm muscle. Furthermore, foci of AChE activity were seen to diminish in size, number, and staining intensity when the rat synaptic basal lamina-enriched preparations were treated with the extraction buffers. In the case of Torpedo, almost all the AChE activity was removed from the pure basal lamina sheets. We therefore conclude that a major portion of extracellular collagen-tailed AChE is extractable from rat and Torpedo synaptic basal lamina by high ionic strength and heparin buffers, although some non-extractable AChE activity remains associated with the junctional regions.The enzyme acetylcholinesterase (AChE) 1 plays a key role in cholinergic neurotransmission (1). Its predominant form at the neuromuscular junction is the collagen-tailed asymmetric form, A 12 , which is located on the extracellular surface positioned for the hydrolysis of acetylcholine. Most of this junctional AChE is associated with the basal lamina (BL), located between the nerve ending and the muscle plasma membrane (2, 3), and can be removed from the cell surface of muscle tissue (4, 5) and mouse myotubes (6) by treatment with collagenase, indicating that the collagenic tail of the enzyme is involved in its anchorage to the BL (7,8). Although the precise mechanisms by which asymmetric AChE forms are anchored to the BL remain elusive (9), there is compelling evidence to suggest that heparan sulfate proteoglycans (HSPGs) or related proteoglycans are involved (7,10). This evidence includes the recent finding that A 12 has two heparin-binding consensus sequences in its collagenic tail (11). Asymmetric AChE forms have a high binding affinity for BL components, particularly HSPGs (12) which are themselves major constituents of basement membranes (13,14). Heparin and heparan sulfate have also been shown to release asymmetric AChE activity from rat muscle end plate regions (15) and BL sheets purified from the electric organ of Discopyge (16). The demonstration that A 12 could bind and be selectively eluted from heparin-agarose columns, whereas non-collagenous forms and A 12 after collagenase treatment could not, proved the direct interaction of A 12 with heparin in vitro (11,17). Direct interactions with heparin/heparan sulfate moieties in vivo have also been demonstrated. Asymmetric AChE forms were shown to bind the surface of HSPGri...

Previous studies have shown that palmitoyl-carnitine is an anti-proliferative agent and a protein kinase C inhibitor. Two new palmitoyl-carnitine analogs were synthesized by replacing the ester bond with a metabolically more stable ether bond. An LD 50 value in the nm range was found in anti-proliferative assays using HL-60 cells and was dependent on the alkyl-chain length. The inhibitory action of these water-soluble compounds on protein kinase C in vitro was greatly increased with respect to palmitoyl-carnitine and was dependent on the length of the alkyl chain. Its effect was mediated by an increase in the enzyme's requirement for phosphatidylserine. Inhibition of the in situ phosphorylation of a physiological platelet protein kinase C substrate and of phorbol ester-induced differentiation of HL-60 cells was also observed. Finally, to test for isoenzyme selectivity, several human recombinant protein kinase C isoforms were used. Only the Ca 2+ -dependent classic protein kinase Cs (a, bI, bII and g) were inhibited by these compounds, yet the activities of casein kinase I, Ca 2+ /calmodulin-dependent kinase and cAMP-dependent protein kinase were unaffected. Thus, these novel inhibitors appear to be both protein kinase C and isozyme selective. They may be useful in assessing the individual roles of protein kinase C isoforms in cell proliferation and tumor development and may be rational candidates for anti-neoplasic drug design.Keywords: anti-proliferative drugs; palmitoyl-carnitine; protein kinase C isoenzymes.Protein kinase C (pkC) isoenzymes play a major role in signal transduction pathways affecting proliferation, differentiation and tumor development [1±4]. In this context, the design of selective pkC inhibitors is of interest because of their potential role as anti-proliferative and anti-neoplasic agents. The classical isoforms of pkC (cpkC-a, cpkC-bI, cpkC-bII and cpkC-g) are activated by Ca 2+ and diacylglycerol or phorbol ester, and phosphatidylserine (PtdSer), whereas the novel isozymes (npkC-d, npkC-:, npkC-u, npkC-h/L and npkC-m) respond to diacylglycerol and phorbol esters but not to Ca 2 . Finally, the atypical isoforms (apkC-z and apkC-zl/i) are activated only by anionic phospholipids such as PtdSer [2]. The various pkC isoforms exhibit marked differences in tissue and subcellular distribution [5], compartmentalization [6], substrate specificity [7,8], protein±protein interactions [9,10] and susceptibility to translocation and down-regulation [11]. These differences are suggestive of functional divergence between individual pkC isotypes, although the exact roles of particular isoforms, particularly in tumor promotion, are as yet unclear, in part because of the lack of pkC-isozyme-selective activators and inhibitors. Although phorbol esters are potent pkC activators, they do not distinguish between individual isoforms and are not selective for pkC because other intracellular receptors for phorbol esters have been identified [12]. Similarly, staurosporine [13], the most potent pkC inhibitor described...

No abstract

Acyl-CoAs are present at high concentrations within the cell, yet are strongly buffered by specific binding proteins in order to maintain a low intracellular unbound acyl-CoA concentration, compatible with their metabolic role, their importance in cell signaling, and as protection from their detergent properties. This intracellular regulation may be disrupted by nonmetabolizables acyl-CoA esters of xenobiotics, such as peroxisome proliferators, which are formed at relatively high concentration within the liver cell. The low molecular mass acyl-CoA binding protein (ACBP) and fatty acyl-CoA binding protein (FABP) have been proposed as the buffering system for fatty acyl-CoAs. Whether these proteins also bind xenobiotic-CoA is not known. Here we have identified new liver cytosolic fatty acyl-CoA and xenobiotic-CoA binding sites as glutathione S-transferase (GST), using fluorescent polarization and a acyl-etheno-CoA derivative of the peroxisome proliferator nafenopin as ligand. Rat liver GST and human liver recombinant GSTA1-1, GSTP1-1 and GSTM1-1 were used. Only class alpha rat liver GST and human GSTA1-1 bind xenobiotic-CoAs and fatty acyl-CoAs, with K d values ranging from 200 nm to 5 mm. One mol of acyl-CoA is bound per mol of dimeric enzyme, and no metabolization or hydrolysis was observed. Binding results in strong inhibition of rat liver GST and human recombinant GSTA1-1 (IC 50 at the nanomolar level for palmitoyl-CoA) but not GSTP1-1 and GSTM1-1. Acyl-CoAs do not interact with the GSTA1-1 substrate binding site, but probably with a different domain. Results suggest that under increased acyl-CoA concentration, as occurs after exposure to peroxisome proliferators, acyl-CoA binding to the abundant class alpha GSTs may result in strong inhibition of xenobiotic detoxification. Analysis of the binding properties of GSTs and other acyl-CoA binding proteins suggest that under increased acyl-CoA concentration GSTs would be responsible for xenobiotic-CoA binding whereas ACBP would preferentially bind fatty acyl-CoAs.

No abstract