Radiation inactivation probe of membrane-bound enzymes: gamma-glutamyltranspeptidase, aminopeptidase N, and sucrase

gamma-Glutamyltranspeptidase (GGT), aminopeptidase N (AP-N), and sucrase in purified rabbit intestinal brush border membrane vesicles were irradiated in situ at -135 degrees C using high energy electrons. Surviving activities of the enzymes were measured as a function of radiation dose, and the functional unit target sizes (corresponding to carbohydrate-free polypeptides) were determined using target analysis. The in situ functional unit sizes were GGT 59 kDa, AP-N 59 kDa, and sucrase 63 kDa. Together with biochemical data determined previously, it is concluded that the noncovalently attached large (approximately 40 kDa) and small (approximately 25 kDa) subunits of GGT are both required for catalytic activity. Furthermore, these data suggest that (i) the membrane-bound form of AP-N consists of one or more noncovalently attached subunits of 59 kDa, each of which is enzymatically active; and (ii) in situ sucrase activity is associated with a subunit of 63 kDa which is noncovalently attached within the sucrase-isomaltase complex.     

Purification and properties of the catalytic domain of human 3-hydroxy-3-methylglutaryl-CoA reductase expressed in Escherichia coli

Three fragments of the cDNA encoding human 3-hydroxy-3-methylglutaryl-CoA reductase, all incorporating the majority of the catalytic domain of the protein, were subcloned into Escherichia coli expression vectors containing the pL promoter. The two larger expressed fragments (58 and 52 kDa) were soluble and had enzymatic activity, while the smallest (48 kDa) was insoluble. The two active fragments were purified by a combination of conventional techniques and affinity chromatography. A number of properties of the two enzymes were compared including specific activity, kinetic parameters, relative solubility, and cold lability. The 52-kDa enzyme was observed to change from a dimeric to monomeric form and to lose activity at 4 degrees C. In contrast, the 58-kDa enzyme was found to be much less cold labile, and was dimeric at both 20 and 4 degrees C. In order to resolve the number of subunits required to form an active site, the number of inhibitor binding sites for a known inhibitor was determined to be one per subunit in the 58-kDa enzyme.     

Homo- and heterotetrameric forms of the membrane-bound metalloendopeptidases meprin A and B

Meprin A and B are disulfide-linked, tetrameric metalloendopeptidases in renal brush border membranes. Meprin A contains 90-kDa subunits (alpha subunits) and is expressed in random-bred and some inbred strains of mice. Meprin B contains subunits of 110 kDa (beta subunits) in situ, and the enzyme from C3H mice, a strain that does not express alpha subunits, has been characterized. Evidence from this and previous studies indicate that beta subunits are expressed in all mouse strains. The tetrameric organization of these meprins was examined in brush border membrane fractions from a random-bred strain (ICR) and two inbred strains of mice (C57BL/6 and C3H/He). Lectin blotting using biotinylated concanavalin A revealed that membranes from the random-bred strain contained three oligomeric complexes of approximately 390, 440, and 490 kDa as determined after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of reducing agents. The subunits in all three oligomers were linked by disulfide bridges. Western blotting using an anti-alpha monoclonal antibody indicated that alpha subunits (90 kDa) were present in the 390- and 440-kDa complexes. Western blotting with a polyclonal antibody specific for beta subunits (110 kDa) indicated the presence of these subunits in the 440- and 490-kDa complexes. Electroelution of the individual oligomers followed by SDS-PAGE under reducing conditions confirmed that the 390- and 490-kDa molecules are homotetramers of alpha and beta subunits, respectively, and that the 440-kDa molecule is a heterotetramer consisting of disulfide-bridged alpha and beta subunits. C57BL/6 mice expressed both alpha and beta subunits and contained tetramers composed of alpha 4 and alpha 2 beta 2. C3H/He mice expressed only the 110-kDa beta subunits and the beta 4 oligomer. This type of multimeric organization of disulfide-linked subunits is unique for the known endopeptidases.     

Protein kinase C in mouse kidney: subcellular distribution and endogenous substrates

The subcellular distribution, kinetic properties, and endogenous substrates of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) were examined in mouse kidney cortex. Protein kinase C associated with the particulate, mitochondrial, and brush border membrane fractions was assayed after solubilization in 0.2% Triton X-100 under conditions shown to be noninhibitory to catalytic activity. Of recovered activity, 52% was associated with the cytosolic fraction; mitochondrial and brush border membrane associated protein kinase C constituted 12 and 3%, respectively, of the activity recovered in the particulate fraction. Protein kinase C associated with brush border membranes exhibited a high affinity for ATP (apparent Km = 62 +/- 10 microM) and the highest apparent maximal velocity (1146 +/- 116 pmol P/(mg protein.min] of the renal fractions examined. Maximal stimulation of protein kinase C by diacylglycerol (in the presence of phosphatidylserine) was achieved at both 25 and 300 microM calcium in all renal fractions. These results are consistent with previous reports demonstrating that diacylglycerol increases the apparent affinity of protein kinase C for calcium. Phorbol 12-myristate 13-acetate, but not 4 alpha-phorbol, was able to substitute for diacylglycerol and stimulate cytosolic and particulate renal protein kinase C. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a specific inhibitor of protein kinase C, led to significant inhibition of catalytic activity in all renal subcellular fractions. Endogenous substrates for protein kinase C were demonstrated in renal cytosolic (26, 45, 63, and 105 kilodaltons (kDa], particulate (26, 33, 68, and 105 kDa), mitochondrial (43 kDa), and brush border membrane (26, 41, 52, 88, and 105 kDa) fractions. The possible physiological significance of protein kinase C in mammalian kidney is discussed.     

Analysis of the effects of snake venom proteinases on the activity of human plasma C1 esterase inhibitor, alpha 1-antichymotrypsin and alpha 2-antiplasmin

Incubation of C1 esterase inhibitor with Crotalid, Viperid and Colubrid snake venoms resulted in enzymatic inactivation of the inhibitor. Intact inhibitor (104 kDa) was converted into an active intermediate species of 89 kDa and then a further cleavage resulted in formation of an 86-kDa inactive inhibitor. In contrast, C1 esterase inhibitor did not lose activity during incubation with Elapid venoms; however, the intact inhibitor was gradually converted to an active species of 89 kDa during the incubation. Human alpha 1-antichymotrypsin was inactivated by all venoms tested, including those from the Elapid family. The 67-kDa intact inhibitor was converted by the venom proteinases to an inactive 63-kDa form. The results suggest that this acute-phase plasma protein is readily susceptible to inactivation by venom proteinases. Human alpha 2-antiplasmin (68 kDa) was cleaved to form a 61-kDa active intermediate, which then underwent a second cleavage to produce an inactive 53-kDa product. Elapid venoms had no effect on alpha 2-antiplasmin activity and did not cleave this inhibitor. All inhibitors were inactivated with catalytic amounts of venom proteinases. No stable proteinase-proteinase inhibitor complexes were detected, and no random proteolysis of the inhibitors occurred.     

Intestinal lactase. Shift in intracellular processing to altered, inactive species in the adult rat

The regulatory mechanism of decline in catalytic activity for intestinal lactase (lactase-phlorizin hydrolase, beta-galactosidase) as mammals mature has not been defined. Solubilized intestinal brush-border membranes from adult male rats (greater than 4 months of age, 200-400 g) were examined by high performance liquid Zorbax GF-450 chromatography, subjected to denaturing acrylamide electrophoresis, blotted to nitrocellulose, and identified by specific polyvalent anti-lactase. Three major species were present within the 235-kDa active lactase peak (225, 130, and 100 kDa). The 100-kDa moiety was also prominent in the approximately 300-kDa region of the GF-450 effluent, suggesting it is a catalytically inactive oligomer. In vivo synthesis and assembly of lactase by intraintestinal pulse [( 35S]methionine, 5 min) and chase (15-120 min) revealed rapid (15 min of chase; maximum, 60 min) intracellular synthesis in the endoplasmic reticulum-Golgi fraction of multiple species (64, 100, 130, 175, and 225 kDa). The 64-kDa species disappeared from the intracellular membrane compartment and was not transferred to the brush-border surface. The 175-kDa moiety appeared to be processed to the 225-kDa unit prior to relocation to the surface membrane. By 120 min, the 100-kDa species became the predominant (approximately 60%) radiolabeled unit in both endoplasmic reticulum-Golgi and brush border. In the adult rat, lactase is assembled in multiple molecular forms that are differentially processed: (a) intracellular degradation (64-kDa unit) or (b) transfer to the brush-border surface as catalytically active (225 and 130 kDa) or inactive (100 kDa) species. Although substantial synthesis of lactase proteins prevails, major changes in processing appear to serve as an important regulatory mechanism producing the maturational decline of catalytic activity. The accompanying article (Castillo, R. O., Reisenauer, A. M., Kwong, L. K., Tsuboi, K. K., Quan, R., and Gray, G. M. (1990) J. Biol. Chem. 265, 15889-15893) extends our studies to synthesis and assembly during the neonatal period of maturation.     

Proteolytic analysis of domain structure in the beta heavy chain of dynein from sea urchin sperm flagella.

The stability of different regions of the beta heavy chain of dynein has been investigated by examining the perturbing effects of methanol, temperature, salt, and nucleotide on the pattern of tryptic digestion. In standard low-salt medium, tryptic proteolysis cleaves the beta heavy chain into three principal polypeptides of 130, 215, and 110 kDa, with the 215-kDa central peptide containing the ATP binding site as well as the vanadate and iron photocleavage sites (Mocz, G., Tang, W.-J. Y., & Gibbons, I. R. (1988) J. Cell Biol. 106, 1607-1614). The 130-kDa peptide is the most stable, and its susceptibility to trypsin appears unaffected by methanol concentrations up to 25% or temperatures up to 45 degrees C, although a 5-kDa region at one end is lost in the presence of salt (greater than 20 mM NaCl). The 215-kDa tryptic peptide contains two regions of different stability: its 123-kDa portion adjoining the 130-kDa peptide is destabilized by mild heat (37 degrees C) or by 25% methanol and becomes digested away to leave the more stable region of 92 kDa that is located toward the 110-kDa peptide and retains the V1 photocleavage site and most of the ATP binding site. The 110-kDa peptide is the least stable and at 37 degrees C, or in the presence of low concentrations of methanol or salt, it rapidly digested to small peptides. The presence of ATP during digestion of the beta heavy chain retards the formation of the 130- and 215-kDa peptides and also protects the 215-kDa peptide from further digestion at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of Bacillus thuringiensis svar. israelensis Cry toxins with binding sites from Aedes aegypti (Diptera: Culicidae) larvae midgut

This work shows in vitro processing of Bacillus thuringiensis svar. isralensis Cry toxins and the capacity of the active fragments to bind the midgut microvilli of Aedes aegypti larvae. Processing of Cry11Aa, Cry4Aa and Cry4Ba yielded double fragments of 38-30, 45-20 and 45-18 kDa, respectively. Competition assays showed that all active (125)I-Cry toxins are able to specifically bind to brush border membrane fractions and they might share a common class of binding sites. The values of IC(50) suggested that toxins do not display high affinity for the receptors from brush border membrane fractions, while dissociation assays showed that binding was irreversible, indicating the insertion of toxins in the cell membrane.     

Localization of the active site and phosphorylation site of Acanthamoeba myosins IA and IB

The 130- and 125-kDa heavy chains of Acanthamoeba myosins IA and IB were radioactively labeled at either the regulatory phosphorylation site or the catalytic site and then subjected to controlled proteolysis by either trypsin or chymotrypsin. The labeled and unlabeled peptides generated during the course of proteolysis were identified by autoradiography and Coomassie Blue staining after separation by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. The relative positions of the phosphorylation and active sites could be deduced. The catalytic site of myosin IA is most probably within 38 kDa of one end of the 130-kDa heavy chain, and the phosphorylation site, which can be no more than 40 kDa away from the catalytic site, would then be between 38 and 78 kDa of that same end of the heavy chain. Possibly, the phosphorylation site is further restricted to the region between 38 and 64 kDa from the end of the heavy chain. The catalytic and phosphorylation sites of myosin IB are both contained within a segment of 62 kDa at one end of the 125-kDa heavy chain and are within 40 kDa of each other. The phosphorylation site may be restricted to a small segment between 60 and 62 kDa from one end of the heavy chain which would limit the possible position of the catalytic site to the region between 20 and 60 kDa of that end.     

Purification of a site-specific endonuclease, I-Sce II, encoded by intron 4 alpha of the mitochondrial coxI gene of Saccharomyces cerevisiae

We have purified to near homogeneity a site-specific, double-stranded DNA endonuclease (I-Sce II) encoded by intron 4 alpha (aI4 alpha) of the yeast mitochondrial coxI gene. Our purification starts with a high salt extract of mitochondria isolated from a yeast strain that overproduces the enzyme because of a block in splicing of aI4 alpha. The final step of purification is an affinity column consisting of covalently bound double-stranded DNA multimers of a synthetic sequence, 5'-TTGGTCATCCAGAAGTAT-3', which contains the I-Sce II cleavage/recognition site. Typical yields of enzyme are 3-5% with a specific activity of approximately 500,000 units/mg, where 1 unit of activity cleaves 50 ng of DNA substrate/h at 30 degrees C. I-Sce II has a monomer molecular mass of 31 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Active enzyme purifies as a 55-kDa species, which we presume to be a homodimer. I-Sce II monomer comigrates with an in vivo synthesized mitochondrial translation product made in the strain that overproduces the enzyme. We conclude that I-Sce II is derived by proteolytic processing of a precursor polypeptide, p62, encoded by an in-frame fusion of coxI exons 1-4 with the downstream aI4 alpha reading frame. I-Sce II is most active at pH 7.5 and at 20-30 degrees C. Endonuclease activity is sensitive to salt and is dependent upon Mg2+ or Mn2+, but is unaffected by inclusion of ATP or GTP. I-Sce II is the first intron-encoded protein to be purified and characterized from yeast mitochondria.     

In vivo processing of Staphylococcus aureus lipase.

The Staphylococcus aureus lipase gene encodes a 76-kDa protein. Extracellular lipase purified from culture supernatants is only 45 to 46 kDa, however. We show that the lipase is secreted in vivo as an 82-kDa protein with full enzymatic activity. It is then sequentially processed, both in culture and in cell-free supernatants, to a mature, 45- to 46-kDa protein. Protein sequencing demonstrates that the N-terminal region of the 82-kDa prolipase, comprising 295 amino acids, is cleaved from the central and C-terminal moieties, which contain the active site. A metallocysteine protease is probably responsible for initiating this processing. The extremely hydrophobic, mature lipase is resistant to further protease degradation and retains the full catalytic activity of the prolipase.     

Localization of the actin-binding sites of Acanthamoeba myosin IB and effect of limited proteolysis on its actin-activated Mg2+-ATPase activity

Acanthamoeba myosin IB contains a 125-kDa heavy chain that has high actin-activated Mg2+-ATPase activity when 1 serine residue is phosphorylated. The heavy chain contains two F-actin-binding sites, one associated with the catalytic site and a second which allows myosin IB to cross-link actin filaments but has no direct effect on catalytic activity. Tryptic digestion of the heavy chain initially produces an NH2-terminal 62-kDa peptide that contains the ATP-binding site and the regulatory phosphorylation site, and a COOH-terminal 68-kDa peptide. F-actin, in the absence of ATP, protects this site and tryptic cleavage then produces an NH2-terminal 80-kDa peptide. Both the 62- and the 80-kDa peptides retain the (NH+4,EDTA)-ATPase activity of native myosin IB and both bind to F-actin in an ATP-sensitive manner. However, only the 80-kDa peptide retains a major portion of the actin-activated Mg2+-ATPase activity. This activity requires phosphorylation of the 80-kDa peptide by myosin I heavy chain kinase but, in contrast to the activity of intact myosin IB, it has a simple, hyperbolic dependence on the concentration of F-actin. Also unlike myosin IB, the 80-kDa peptide cannot cross-link F-actin filaments indicating the presence of only a single actin-binding site. These results allow the assignment of the actin-binding site involved in catalytic activity to the region near, and possibly on both sides of, the tryptic cleavage site 62 kDa from the NH2 terminus, and the second actin-binding site to the COOH-terminal 45-kDa domain. Thus, the NH2-terminal 80 kDa of the myosin IB heavy chain is functionally similar to the 93-kDa subfragment 1 of muscle myosin and most likely has a similar organization of functional domains.     

Regulation of squid visual phospholipase C by activated G-protein alpha.

Phospholipase C (PLC) is the key enzyme in the phototransduction cascade of invertebrate rhabdomeric photoreceptors. In addition to 130 kDa PLC, a 95 kDa protein recognized by antibody against the catalytic site of PLC was found in the squid retina. The PLC-like 95 kDa protein (95 kDa PLC) was produced from 130 kDa PLC by an intrinsic protease in the presence of calcium. The 130 kDa PLC was stimulated by the active form of Gq-class G-protein alpha (Gq alpha), but the 95 kDa PLC was not, although their PLC activity was similar. A 35 kDa fragment, the counterpart of 95 kDa PLC, was not recognized by antibodies against catalytic site or N-terminal site of the 130 kDa PLC, indicating that the cleavage site is on the C-terminal side beyond the catalytic site. In the presence of a large excess of the 35 kDa fragment, 95 kDa PLC was stimulated by Gq alpha to a similar extent as intact 130 kDa PLC. These results indicate that the C-terminal polypeptide of PLC is necessary for regulation of its enzyme activity by Gq alpha. The uncoupling of PLC from Gq alpha, caused by limited proteolysis, is therefore a candidate regulatory mechanism of the phototransduction cascade in rhabdomeric photoreceptors.     

Development of the small intestine in the hypophysectomized rat. II. Influence of cortisone, thyroxine, growth hormone, and prolactin.

The intestinal deficiencies caused by hypophysectomy of rats at 6 days of age can be repaired to varying degrees by thyroxine or cortisone but not by growth hormone or prolactin. Administration of daily doses of thyroxine alone from 19–22 days raises duodenal alkaline phosphatase activity to normal levels at 24 days; it has a strong effect on jejunal sucrase and maltase, although these activities remain below those of controls. Thyroxine causes a marked increase in rough endoplasmic reticulum and restores the Golgi complexes to their normal appearance. It also elicits an intensification of periodic acid-Schiff (PAS) stainability of the brush border. Cortisone acetate given from 19 to 22 days elevates sucrase and maltase to normal levels but does not fully restore phosphatase activity. Like thyroxine, cortisone causes intensification of PAS staining of the brush border and also increases rough endoplasmic reticulum. It seems to stimulate Golgi activity, but results in the appearance of a variety of abnormal forms. The defects in Golgi configuration, brush border carbohydrate content, and activity of glycoprotein enzymes that are bound to the brush border may all reflect impaired glycosylation in the hypophyseoprivic state; the results of thyroxine or cortisone administration suggest that both hormones may affect glycosylation but in different ways.     

Affinity labeling the DNA polymerase alpha complex. Identification of subunits containing the DNA polymerase active site and an important regulatory nucleotide-binding site

Pyridoxal 5'-phosphate (PLP) inhibits DNA polymerase activity of the intact multifunctional DNA polymerase alpha complex by binding at either of two sites which can be distinguished on the basis of differential substrate protection. One site (PLP site 1) corresponds to an important nucleotide-binding site which is distinct from the DNA polymerase active site and which appears to correspond to the DNA primase active site while the second site (PLP site 2) corresponds to the dNTP binding domain of the DNA polymerase active site. A method for the enzymatic synthesis of high specific activity [32P]PLP is described and this labeled PLP was used to identify the binding sites described above. PLP inhibition of DNA polymerase alpha activity was shown to involve the binding of only a few (one to two) molecules of PLP/molecule of DNA polymerase alpha, and this label is primarily found on the 148- and 46-kDa subunits although the 63-, 58-, and 49-kDa subunits are labeled to a lesser extent. Labeling of the 46-kDa subunit by [32P]PLP is the only labeling on the enzyme which is blocked or even diminished in the presence of nucleotide alone, and, therefore, this 46-kDa subunit contains PLP site 1. Labeling of the 148-kDa subunit is enhanced in the presence of template-primer, suggesting that this subunit undergoes a conformational change upon binding template-primer. Furthermore, labeling of the 148-kDa subunit is the only labeling on the enzyme which can be specifically blocked only by the binding of both template-primer and the correct dNTP in a stable ternary complex. Therefore, the 148-kDa subunit contains PLP site 2, which corresponds to the dNTP binding domain of the DNA polymerase active site.     

Conformational transitions in the myosin head induced by temperature, nucleotide and actin. Studies on subfragment-1 of myosins from rabbit and frog fast skeletal muscle with a limited proteolysis method

Tryptic digestion patterns reveal a close similarity of the substructure of frog subfragment-1 (S1) to that established for rabbit S1. The 97-kDa heavy chain of chymotryptic S1 of frog myosin is preferentially cleaved into three fragments with apparent molecular masses of 29 kDa, 49 kDa and 20 kDa. These fragments correspond to the 27-kDa, 50-kDa and 20-kDa fragments of rabbit S1, respectively; this is indicated by the sequence of their appearance during digestion, by the suppression by actin of the generation of the 49-kDa and 20-kDa peptides, and by a nucleotide-promoted cleavage of the 29-kDa peptide to a 24-kDa fragment and the 49-kDa peptide to a 44-kDa fragment, analogous to the nucleotide-promoted cleavage of the 27-kDa and 50-kDa fragments of rabbit S1 to the 22-kDa and 45-kDa peptides. The same changes in the digestion patterns as those produced by the presence of nucleotide (ATP or its beta,gamma-imido analog AdoP P[NH]P) at 25 degrees C were observed when the digestion was carried out at 0 degrees C in the absence of nucleotide. The low-temperature-induced changes were particularly well seen in the preparations from frog myosin. The presence of ATP or AdoP P[NH]P at 0 degrees C enhanced, whereas the complex formation with actin prevented, the low-temperature-induced changes. The results are consistent with there being two fundamental conformational states of the myosin head in an equilibrium that is dependent on the temperature, the nucleotide bound at the active site, and the presence or absence of actin.     

Phosphorylation of brush border myosin I by protein kinase C is regulated by Ca(2+)-stimulated binding of myosin I to phosphatidylserine concerted with calmodulin dissociation.

Brush border myosin I from chicken intestine is phosphorylated in vitro by chicken intestinal epithelial cell protein kinase C. Phosphorylation on serine and threonine to a maximum of 0.93 mol of P/mol of myosin I occurs within an approximately 20 kDa region at the end of the COOH-terminal tail of the 119-kDa heavy chain. The effects of Ca2+ on myosin I phosphorylation by protein kinase C are complex, with up to 4-fold stimulation occurring at 0.5-3 microM Ca2+, and up to 80% inhibition occurring at 3-320 microM Ca2+. Phosphorylation required that brush border myosin I be in its phosphatidylserine vesicle-bound state. Previously unknown Ca2+ stimulation of brush border myosin I binding to phosphatidylserine vesicles was found to coincide with Ca2+ stimulation of phosphorylation. A myosin I proteolytic fragment lacking approximately 20 kDa of its tail retained Ca(2+)-stimulated binding, but showed reduced Ca(2+)-independent binding. Ca(2+)-dependent phosphatidylserine binding is apparently due to the concomitant phosphatidylserine-promoted, Ca(2+)-induced dissociation of up to three of the four calmodulin light chains from myosin I. Four highly basic putative calmodulin-binding sites in the Ca(2+)-dependent phosphatidylserine binding region of the heavy chain were identified based on the similarity in their sequence to the calmodulin- and phosphatidylserine-binding site of neuromodulin. Calmodulin dissociation is now shown to occur in the low micromolar Ca2+ concentration range and may regulate the association of brush border myosin I with membranes and its phosphorylation by protein kinase C.     

Molecular cloning of two new human paralogs of 85-kDa cytosolic phospholipase A2

Two new cloned human cDNAs encode paralogs of the 85-kDa cytosolic phospholipase A2 (cPLA2). We propose to call these cPLA2beta (114 kDa) and cPLA2gamma (61 kDa), giving the name cPLA2alpha to the well known 85-kDa enzyme. cPLA2beta mRNA is expressed more highly in cerebellum and pancreas and cPLA2gamma more highly in cardiac and skeletal muscle. Sequence-tagged site mapping places cPLA2beta on chromosome 15 in a region near a phosphoinositol bisphosphate phosphatase. The mRNA for cPLA2beta is spliced only at a very low level, and Northern blots in 24 tissues show exclusively the unspliced form. cPLA2beta has much lower activity on 2-arachidonoyl-phosphatidylcholine liposomes than either of the other two enzymes. Its sequence contains a histidine motif characteristic of the catalytic center of caspase proteases of the apoptotic cascade but no region characteristic of the catalytic cysteine. Sequence-tagged site mapping places cPLA2gamma on chromosome 19 near calmodulin. cPLA2gamma lacks the C2 domain, which gives cPLA2alpha its Ca2+ sensitivity, and accordingly cPLA2gamma has no dependence upon calcium, although cPLA2beta does. cPLA2gamma contains a prenyl group-binding site motif and appears to be largely membrane-bound. cPLA2alpha residues activated by phosphorylation do not appear to be well conserved in either new enzyme. In contrast, all three previously known catalytic residues, as well as one additional essential arginine, Arg-566 in cPLA2alpha, are conserved in both new enzyme sequences. Mutagenesis shows strong dependence on these residues for catalytic activity of all three enzymes.     

Affinity labelling with MgATP analogues reveals coexisting Na+ and K+ forms of the alpha-subunits of Na+/K+-ATPase.

To test the hypothesis that Na+/K+-ATPase works as an (alpha beta)2-diprotomer with interacting catalytic alpha-subunits, tryptic digestion of pig kidney enzyme, that had been inactivated with substitution-inert MgATP complex analogues, was performed. This led to the demonstration of coexisting C-terminal Na+-like 80-kDa as well as K+-like 60-kDa peptides and N-terminal 40-kDa peptides of the alpha-subunit. To localize the ATP binding sites on tryptic peptides, studies with radioactive MgATP complex analogues were performed: Co(NH3)4-8-N3-ATP specifically modified the E2ATP (low affinity) binding site of Na+/K+-ATPase with an inactivation rate constant (k2) of 12 x 10-3.min-1 at 37 degrees C and a dissociation constant (Kd) of 207 +/- 28 microm. Tryptic digestion of the [gamma32P]Co(NH3)4-8-N3-ATP-inactivated and photolabelled alpha-subunit (Mr = 100 kDa) led, in the absence of univalent cations, to a K+-like C-terminal 60-kDa fragment which was labelled in addition to an unlabelled Na+-like C-terminal 80-kDa fragment. Tryptic digestion of [alpha32P]-or [gamma32P]Cr(H2O)4ATP - bound to the E1ATP (high affinity) site - led to the labelling of a Na+-like 80-kDa fragment besides the immediate formation of an unlabelled K+-like N-terminal 40-kDa fragment and a C-terminal 60-kDa fragment. Because a labelled Na+-like 80-kDa fragment cannot result from an unlabelled K+-like 60-kDa fragment, and because unlabelled alpha-subunits did not show any catalytic activity, the findings are consistent with a situation in which Na+- and K+-like conformations are stabilized by tight binding of substitution-inert MgATP complex analogues to the E1ATP and E2ATP sites. Hence, all data are consistent with the hypothesis that ATP binding induces coexisting Na+ and K+ conformations within an (alphabeta)2-diprotomeric Na+/K+-ATPase.     

Translocation of alpha subunits of stimulatory guanine nucleotide-binding proteins through stimulation of the prostacyclin receptor in mouse mastocytoma cells.

The translocation of the alpha subunits of Gs from the membrane to the cytosol by iloprost, a stable prostacyclin analogue, was studied in mouse mastocytoma P-815 cells. In the presence of guanosine 5'-O-(thiotriphosphate) (GTP gamma S), iloprost stimulated the adenylate cyclase activity, caused the release of both 42- and 45-kDa proteins reactive with the anti Gs alpha carboxyl-terminal antibody, RM/1, from the membrane and attenuated cholera toxin-catalyzed ADP-ribosylation of the 42- and 45-kDa proteins in the membrane. The iloprost-stimulated adenylate cyclase activity and release of Gs alpha from the membrane were markedly suppressed by RM/1. Cholera toxin treatment also stimulated the adenylate cyclase activity and release of Gs alpha from the membrane, and iloprost synergistically potentiated these actions of cholera toxin. In mastocytoma cells, iloprost induced the translocation of both 42- and 45-kDa Gs alpha from the membrane to the cytosol, 45-kDa Gs alpha remaining in the cytosol for a longer time than 42- kDa Gs alpha. Whereas 42-kDa Gs alpha in the cytosol was eluted at the position of Mr = approximately 40,000 45-kDa Gs alpha was eluted at the position of Mr = approximately 120,000 from a Superose 12 gel filtration column. In contrast, both 42- and 45-kDa Gs alpha released in vitro from the membrane by iloprost plus GTP gamma S were eluted at the position of Mr = approximately 40,000, but only 45-kDa Gs alpha was eluted at the position of Mr = approximately 120,000 when it was incubated with cytosol. These results taken together demonstrate that iloprost induces the translocation of both 42- and 45-kDa Gs alpha from the membrane to the cytosol and that only the 45-kDa Gs alpha released exists in the cytosol as a soluble complex with unidentified component(s) in mastocytoma cells.