A recombinant form of the catalytic subunit of phosphorylase kinase that is soluble, monomeric, and includes key C-terminal residues.

Residues 302-326 of the catalytic (gamma) subunit of phosphorylase kinase (PhK) may comprise an autoinhibitory, pseudosubstrate domain that binds calmodulin. To study this, the cDNA corresponding to rabbit muscle PhKgamma was expressed using Escherichia coli. This yielded two stable, high-activity PhKgamma forms (35 and 42 kDa by SDS-PAGE) that were smaller than an authentic sample of rabbit muscle PhKgamma (45 kDa by SDS-PAGE). Each recombinant form was purified to homogeneity. The N-terminal sequence of the larger, 42-kDa form (pk42) matched that of the rabbit muscle enzyme. This suggested that pk42 consisted of PhKgamma residues 1-362, including the putative calmodulin-binding, autoinhibitory domain. Kinetic parameters obtained for pk42 were like those previously reported for the intact gamma subunit. This implied that the lack of 25 PhKgamma C-terminal residues did not affect phosphorylase kinase activity, but greatly improved enzyme stability. An additional 60 residues were removed from the C-terminus of pk42 using the protease m-calpain. This increased the kinase activity 1.5-fold. Consistent with this, the activity of a mutant PhKgamma that consisted of residues 1-300, denoted gamma1-300, was like that of the m-calpain-treated enzyme. Therefore, although the effect was small, some influence by the C-terminus of pk42 was noted. Moreover, when pk42 was incubated with ATP alone, a C-terminal threonine residue became phosphorylated. Although the influence of this autophosphorylation cannot be inferred from this data, it was evidence that the C-terminus accessed the enzyme's active site. Taken together, these data imply that pk42 will be useful to study phosphorylase kinase structure/activity relationships.

Purification of a lysophospholipase from bovine brain that selectively deacylates arachidonoyl-substituted lysophosphatidylcholine.

A high activity lysophospholipase A (lysoPLA) was purified from the soluble fraction of bovine brain. The separation included sequential DEAE-Sephacel, phenyl-Sepharose FF, heparin-Sepharose CL-6B, and Q-Sepharose FF column chromatography. Mono Q, Sephacryl S300HR, and hydroxylapatite column chromatography in the presence of the detergent CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate) and glycerol further purified the activity to 17,000-fold. The enzyme was purified to homogeneity by polyacrylamide gel electrophoresis using nondenaturing conditions. The pure enzyme migrated as a single polypeptide of 95 kDa mass by SDS-polyacrylamide gel electrophoresis and deacylated arachidonoyl-lysophosphatidylcholine (ara-lysoPC) at rate of 70 micromol/(min mg). The enzyme showed selectivity for arachidonoyl-substituted lysoPC, since palmitoyl-lysoPC was deacylated at a much lower rate (7 micromol/(min mg)). LysoPLA activity was maximal at pH 7.4-8.0 and was increased 1.3-fold by MgCl2 (5 mM). By including MgCl2, however, the range of optimal activity was expanded to pH values up to 9.0. The 95-kDa protein also deacylated arachidonoyl groups from 1-O-hexadecyl-2-arachidonoyl-PC (PLA2 activity) at a rate of 15 micromol/(min mg). Moreover, the deacylation of arachidonoyl groups from diacylPC was greatly increased by including purified bovine brain PLA1 in the reaction mixture. Thus, the same 95-kDa polypeptide catalyzed both lysoPLA and PLA2 activities, but the rate of arachidonoyl group deacylation was increased by prior sn-1 deacylation. Finally, the 95-kDa polypeptide cross-reacted with antibodies raised against a human recombinant cPLA2, implying that the 95-kDa protein is structurally similar to cPLA2. Additionally, these data suggest that the combined actions of PLA1 and the 95-kDa protein generate significant amounts of free arachidonic acid in the brain.

Subcellular fractions of bovine brain degrade phosphatidylcholine by sequential deacylation of the sn-1 and sn-2 positions.

Phosphatidylcholine (PC) metabolism was investigated using cytosol (fraction I) and particulate fractions of bovine brain that were enriched with microsomes (fraction II), plasma membranes (fraction III) or mitochondria (fraction IV). Fractions I-III incubated with 1-palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphocholine yielded [14C]arachidonic acid at near equal rates, whereas only fraction I accumulated significant amounts of 2-[14C]arachidonoyl-sn-glycero-3-phosphocholine. Much slower rates of arachidonic acid release were observed using an ether PC (1-O-hexadecyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine). Moreover, arachidonic acid yield from the diacyl, but not ether PC was slowed by pretreating fractions I-III, but not IV, with phenylmethylsulfonyl fluoride (PMSF). Coincident with this decreased arachidonic acid, 2-[14C]arachidonoyl-sn-glycero-3-phosphocholine was increased, indicating high PLA1 activity. Taken together these data suggest that arachidonic release was largely dependent on initial deacylation of position sn-1. Incubating each untreated fraction with 2-[3-H]arachidonoyl-sn-glycero-3-phosphocholine yielded [3H]arachidonic acid (lysophospholipase A2 activity) at rate that was substantially greater than that using the comparable PMSF-treated fraction. Thus, the large effect of PMSF on arachidonic acid release can be accounted for if much of the fatty acid formation arose from the sequential sn-1 and sn-2 deacylation of diacyl-PC by phospholipase A1 and lysophospholipase A2. When PMSF-treated fractions were incubated with 2-[3H]arachidonoyl-sn-glycero-3-phosphocholine, [3H]PC accumulated at low rates that were enhanced by adding coenzyme A or stearoyl-coenzyme A. Thus, the lysophospholipid was also reacylated to form PC, but this reaction was negligible in the absence of PMSF and added cofactors. In summary, we conclude that, in brain subcellular fractions, deacylation of the sn-1 position of diacyl-PC proceeded more rapidly than sn-2 hydrolysis. There was substantial further metabolism of 2-acyl lysophospholipids due to the combined activities of a PMSF-sensitive and -insensitive lysophospholipase. Finally, the sequential deacylation of diacyl-PC by phospholipase A1 and lysophospholipase A2 probably accounted for the major portion of arachidonic acid produced.

Phospholipid interactions affect substrate hydrolysis by bovine brain phospholipase A1.

The specificity of substrate hydrolysis by bovine brain phospholipase A1 (PLA1) was examined. In the presence of Mg2+, using pH values of 7 to 9, the purified enzyme deacylated 1-palmitoyl-2-[1-14C]arachidonoyl-phosphatidylethanolamine yielding 2-[1-14C]arachidonoyl-lysophosphatidylethanolamine at a rate of 70 mumol/min per mg. In the absence of Mg2+, however, the reaction rate slowed at pH values above 7.25. In contrast, brain PLA1 slowly (3.8 mumol/min per mg) hydrolyzed 1-palmitoyl-2-[1-14C]arachidonoyl-phosphatidylcholine (PAPC) unless phosphatidylserine (PS) was included. Maximal PAPC hydrolyzing activity required a PAPC/PS molar ratio of 2.5:1, Mg2+, and a pH value of 8.5-9.5. Replacing PS with phosphatidylethanolamine (PE) or phosphatidic acid (PA), but not phosphatidylinositol (PI), produced a similar effect. Moreover, hydrolysis of either arachidonoyl-substituted or dipalmitoyl-substituted PC at pH 7.5 was enhanced by increasing the mol fraction of PE. Brain PLA1 also hydrolyzed 1-stearoyl-[1-14C]arachidonoyl-PI with high velocity, but only if the substrate was dispersed in PE vesicles. In contrast, the velocity of PS, 1-palmitoyl-lyso-PC or diacylglycerol hydrolysis was low and unaffected by PE. In summary, PLA1 hydrolyzed PE with high velocity and specificity, whereas a high rate of PC or PI hydrolysis was observed only if PS, PE, or PA was present. In addition, PLA1 activity was greatly influenced by pH and Mg2+, implying that the substrate conformation is important to the catalytic efficiency of PLA1. Finally, the high rate of PE, PC or PI hydrolysis suggests PLA1 significantly contributes to the turnover of these phospholipids in the brain.

Purification and properties of phospholipase A1 from bovine brain.

Phospholipase A1 (PLA1) was isolated from a soluble fraction of bovine brain. The purification included sequential DEAE-Sephacel, phenyl-Sepharose FF, and heparin-Sepharose CL-6B column chromatography. Mono Q, Sephacryl S-300, and Mono S high resolution column chromatography in the presence of the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (10 mM) and glycerol (10%, v/v) was required to further separate the enzyme from contaminating material. The purified PLA1 eluted from the Sephacryl S-300HR column in a volume corresponding to a molecular mass of 365 kDa and migrated as two bands (M(r) = 112,000 and 95,000) when separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Chromatofocusing, hydroxylapatite, and lectin affinity column chromatography and nondenaturing polyacrylamide gel electrophoresis were unsuccessful in separating the two electrophoretic bands, implying a close association or similarity. The purified enzyme was stable in solutions containing detergent and glycerol and was insensitive to metal chelators, dithiothreitol, phenylmethylsulfonyl fluoride, and diisopropyl fluorophosphate, but was inactivated by heat (60 degrees C) and ZnCl2. At pH 7.5, the purified enzyme showed highest specific activity, 23.8 mumol/min-mg, when 1-palmitoyl-2-[1-14C]arachidonoyl-phosphatidylethanolamine was the substrate. The rate of catalysis was optimal at a pH of 9.0 and could be enhanced 2-fold by Ca2+, Mg2+, and Sr2+, but not Mn2+. The enzyme catalyzed the specific hydrolysis of acyl groups from the sn-1 position of a broad range of phospholipid substrates, including lysophospholipids, and accounts for most of the soluble phospholipase A1 activity of bovine brain.

Isolation and primary structure of a potent toxin from the venom of the scorpion Centruroides sculpturatus Ewing.

A potent toxin has been purified from the venom of the scorpion Centruroides sculpturatus Ewing using the ion-exchange resin CM-Sepharose CL-6B at basic pH. The toxin, designated CsE M1, comprised 65 amino acid residues and its primary structure was established as: Lys-Glu-Gly-Tyr-Leu-Val-Asn-Ser-Tyr-Thr10-Gly-Cys-Lys-Tyr-Glu-Cys- Leu-Lys-Leu- Gly20-Asp-Asn-Asp-Tyr-Cys-Leu-Arg-Glu-Cys-Arg30-Gln-Gln-Tyr- Gly-Lys-Ser-Gly-Gly - Tyr-Cys40-Tyr-Ala-Phe-Ala-Cys-Trp-Cys-Thr-His-Leu50-Tyr-Glu- Gln-Ala-Val-Val-Trp - Pro-Leu-Pro60-Asn-Lys-Thr-Cys-Asn. CsE M1 is the most lethal protein to be identified in C. sculpturatus venom and the LD50 of the toxin, determined by subcutaneous injection into Swiss mice, is 87 micrograms/kg. CsE M1 shows strong structural similarity (92% positional identity) to the most potent beta-toxin, Css II, from the Mexican scorpion, Centruroides suffusus suffusus but is quite dissimilar to the previously characterized toxins with low potency isolated from C. sculpturatus Ewing.

Structural studies on porcine hemopexin.

1. Porcine hemopexin was isolated from the serum of a single animal and purified to homogeneity. 2. Porcine hemopexin has an apparent Mw of 67,000, binds heme in a 1:1 molar ratio and consists of 24% N-linked oligosaccharides. The amino acid composition of porcine hemopexin compares well with the amino acid composition of human and rabbit hemopexins. 3. Limited tryptic hydrolysis of apohemopexin generates stable peptides of apparent Mw 42,000, 25,000, 24,000 and 21,000. The tryptic peptide of apparent Mw 42,000 (peptide I) binds heme in a 1:1 molar ratio, consists of 33% N-linked oligosaccharides and is derived from the amino terminal of intact hemopexin. The three peptides of smaller-Mw (collectively peptide II) represent the carboxyl terminal half of hemopexin, do not contain N-linked oligosaccharides and have no heme-binding capability. The Mw heterogeneity of peptide II is likely due to cleavage at secondary sites. 4. Under nondissociating electrophoresis two bands are resolved for hemopexin and peptide I, indicating the possibility of polymorphism in porcine hemopexin.

Bovine serum hemopexin: properties of the protein from a single animal.

1. Hemopexin was isolated from bovine serum of a single animal in a yield of 0.5 mg/ml. 2. Bovine hemopexin was found to exist in two isoforms of mol. wt 68,000 and 65,000. 3. Treatment of hemopexin with glycopeptidase F yields a single band corresponding to a mol. wt of 51,000. 4. The protein binds heme on an equimolar ratio and shows a single component in reverse-phase high performance liquid chromatography. 5. The amino acid composition of bovine hemopexin compares with that of hemopexin isolated form other animals.