Regulation of high molecular weight bovine brain neutral protease by phospholipids in vitro.

The activity of the heat stable, glycosylated high molecular weight bovine brain neutral protease (HMW protease) is differentially regulated by phospholipids. While phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidic acid (PA) had only marginal stimulatory effect (40-75%) on the activity of HMW protease, lysophoshatidylcholine (lysoPC) and lysophosphatidic acid (lysoPA) activated the enzyme by more than two-fold. Both lysoPC and lysoPA exhibited concentration-dependent saturation kinetics for the activation of HMW protease. Surprisingly, phosphoinositides (phosphatidylinositol, PI; phosphatidylinositol 4-phosphate, PIP; and phosphatidylinositol 4,5-bisphosphate, PIP2) modulated the activity of protease differently: activation of the enzyme was higher with PIP (90%) as compared to PI (21%), whereas PIP2 inhibited the enzyme (16%). The inhibition of the protease by PIP2 was concentration-dependent. During receptor-coupled cell activation, phospholipase A2 (PLA2) converts PC and PA to lysoPC and lysoPA, respectively; PI is converted to PIP2 by successive enzymatic phosphorylation by PI 4-kinase and PIP 5-kinase; and phospholipase C (PLC) degrades PIP2 to diacylglycerol and inositol 1,4,5-trisphosphate. Therefore, the data suggest that HMW protease may be coupled to cell signal transduction where PLA2, PI 4-kinase, PIP 5-kinase and PLC are involved.

Immunocytochemical localization of the high molecular weight protease in brain.

Polyclonal antiserum against a high molecular weight glycosylated protease, purified from calf brain cytosol, was raised in rabbit and purified by immunoaffinity. The antibody specifically immunoreacted with the M(r) = 165,000 and 155,000 polypeptides of the protease. Immunocytochemical localization data revealed that the protease is localized in the pyramidal neurons, granular and glial cells of the hippocampus. Microscopic analysis of the pyramidal neurons indicates that the protease is present in the cytoplasm and extends to the dendrite and axon. The nuclei of these neurons remain unstained.

High molecular weight glycosylated protease in calf brain. Purification and partial characterization.

A high molecular weight protease has been purified to homogeneity from calf brain cytosol. The purification procedure involves ammonium sulfate fractionation of the cytosol followed by chromatography on DEAE-Sephacel, hydroxylapatite, concanavalin A-Sepharose 4B and Sephacryl S-300. The molecular weight of the native protease was estimated to be Mr = 465,000 by high pressure liquid chromatography. It is composed of a closely moving doublet of Mr = 165,000 and 155,000, as determined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It degrades [methyl-14C] alpha-casein with a broad pH optimum of 6.8-8.5. [methyl-14C]bovine serum albumin and 125I-bovine serum albumin are hydrolyzed to the same extent as [methyl-14C]alpha-casein, whereas [methyl-14C]methemoglobin is hydrolyzed to half the extent of [methyl-14C] alpha-casein. Divalent cations, nucleotides, and known protease inhibitors (phenylmethylsulfonyl fluoride, p-chloromercuribenzoate, iodoacetic acid, N-ethylmaleimide, leupeptin, antipain, pepstatin, and hemin) have no effect on the activity of the protease. The protease is glycosylated and appears to aggregate readily. Aggregation may be reversed by treating the protease with certain organic solvents. The protease seems to maintain full activity after heat treatment. Electron microscopic data reveals a spherical structure of 20-nm diameter.

Third form of calcium-activated neutral proteinase from calf brain: purification, partial characterization and comparison of properties with other forms.

A third form (CANP3) of calcium-activated neutral proteinase (CANP) has been purified, 3900-fold, to near homogeneity from calf brain cortex. The purification procedure is based on the one recently developed for the purification of CANP1 and CANP2. The molecular weight of CANP3, as judged on SDS-polyacrylamide gel electrophoresis was Mr 78,000. A protein with an apparent Mr 17,000 co-purified with the proteinase. At neutral pH (7.2), it was maximally active at 260 microM CaCl2. In the presence of CaCl2, CANP1 and CANP3 were autolyzed very rapidly, whereas the autolysis of CANP2 was slow and gradual. The autolyzed CANP1 and CANP3 responded differently to CaCl2; CANP1 lost activity completely, whereas CANP3 was fully active at 0.5 microM CaCl2. Despite the opposite behavior of these proteinases in the presence of Ca2+, no significant differences in the peptide maps of the three proteinases were observed. Neurofilaments, neurotubules and myelin basic protein (MBP) were degraded by each of the proteinases. Monoclonal antibodies raised against CANP2 reacted almost equally with CANP1 and CANP3. As with CANP1 and CANP2, leupeptin and sulfhydryl-modifying compounds, NEM and iodoacetic acid, inhibited the activity of CANP3.

Micromolar Ca2+ requiring protease from human platelets: purification, partial characterization and effect on the cytoskeletal proteins.

A calcium-activated neutral protease (CANP) has been purified 2,800 fold, to near homogeneity, from human platelets. The purification procedure involved ammonium sulfate fractionation of the platelet cytosol followed by chromatography on Sephacryl S-200, DEAE-Sephacel, Agarose-Hexylamine, Agarose-Octylamine and alpha-casein-Sepharose 4B affinity gel. The protease consisted of two polypeptides of Mr = 74,000 and 28,000 as judged on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It hydrolyzed [methyl-14C] alpha-casein at a significant rate of 37 degrees C which was, therefore, used as an exogenous substrate. Microtubules and intermediate filament proteins were also susceptible to hydrolysis by the purified protease. It attained maximum activity at 0.06 uM CaCl2 and displayed two pH maxima: one at 5.5 and the other at 6.5. The protease was fully active in the presence of MnCl2 and was about 75% active with BaCl2 and SrCl2. Among the actinomycete protease inhibitors, leupeptin, antipain and pepstatin, the order of inhibition was: leupeptin greater than antipain greater than pepstatin. The protease was also inhibited by sulfhydryl modifying agents.

Purification and degradation of purified neurofilament proteins by the brain calcium-activated neutral proteases.

The effect of the three forms (CANP1, CANP2 & CANP3) of calf brain calcium activated neutral protease (CANP) on the hydrolysis of purified neurofilament triplet proteins was investigated. It was observed that: each of the purified neurofilament proteins, was hydrolyzed slowly by CANP2 whereas the hydrolysis of 150 KDa and 68 kDa proteins by CANP1 & CANP3 was rapid; when assembled neurofilaments were used as a substrate, again differences in the rate and extent of degradation of the triplet proteins by the three proteases were observed. For example, little cleavage of the 68 kDa protein by CANP2 and CANP3 was noted whereas 210 kDa and 150 kDa proteins remained largely intact. CANP1 degraded the 150 kDa and 68 kDa proteins more rapidly than 210 kDa protein, where only a slight effect was noted. These data provide further proof of the existence of three different forms of CANP in the brain, and indications of the resistance of 210 kDa protein to proteolysis which may be compatible with its proposed special role in crossbridge formation.

Purification and partial characterization of two forms of Ca2+-activated neutral protease from calf brain synaptosomes and spinal cord.

Two forms ( CANP1 and CANP2 ) of a calcium activated neutral protease (CANP) have been purified to near homogeneity from calf brain synaptosomes and spinal cord. The procedure involves ammonium sulfate fractionation of the brain synaptosome or spinal cord cytosol followed by chromatography on DEAE-Sephacel, Hydroxylapatite and alpha-casein-CH-Sepharose 4B affinity gel. The molecular mass of each of the proteases is 78,000 as judged on SDS-PAGE. A protein with apparent molecular mass of 17,000 copurifies with each of the proteases. CANP1 was maximally active at 600 microM while CANP2 exhibited maximum activity at about 2 microM Ca2+. Both of the proteases were inhibited by sulfhydryl modifying agents and leupeptin.

Purification and characterization of two forms of Ca2+-activated neutral protease from calf brain.

Two forms (CANP1 and CANP2) of a calcium-activated neutral protease (CANP) have been purified, 1,950- and 1,250-fold, respectively, to near homogeneity from calf brain. The purification procedure involves ammonium sulfate fractionation of the brain cytosol followed by chromatography on DEAE-Sephacel, hydroxylapatite, and alpha-casein-CH-Sepharose 4B affinity gel. A protein with apparent Mr = 17,000 copurifies with each of the proteases. This protein was separated by chromatography on a reactive red-120 agarose. Preliminary experiments indicate that, in the absence of this protein, the activity of each of the proteases was reduced. These observations raise the possibility that the 17,000-Da protein may regulate the activity of these proteases. Each of the proteases have similar apparent Mr = 78,000 as judged on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Except for casein, hemocyanin, and hemoglobin, no other exogenous proteins tested were significantly hydrolyzed by either of the proteases. [ methyl-14C ]alpha-Casein or methemoglobin was routinely used as a substrate for both of the enzymes. The endogenous proteins, neurotubules (microtubule-associated proteins and tubulin), neurofilament triplet proteins and desmin from smooth muscle were extensively hydrolyzed by both of the proteases. A marked difference was found in their requirement for CaCl2. CANP1 was maximally active at 700 microM while CANP2 exhibited highest activity at 2 microM CaCl2. Both displayed maximum activity at pH 7.5, although the overall pH profiles were slightly different. Among the actinomycete protease inhibitors, antipain, leupeptin, and pepstatin, leupeptin was highly effective in inhibiting the activities of both enzymes. Both of the proteases were also inhibited by sulfhydryl modifying agents. Metal ions other than CaCl2 were poor activators of the activity of either protease.

Purification and characterization of myosin from calf brain.

Actomyosin complex was extracted from the brain cortex in a medium consisting of low salt, ATP, and EDTA, in the presence of protease inhibitors, followed by ammonium sulfate fractionation. Myosin was then purified from the actomyosin. Myosin obtained according to the procedure used was significantly contaminated with actin high (greater than 200,000 dalton) and low molecular weight proteins. Therefore, an alternative method based on affinity chromatography (Blue Dextran/Sepharose) and gel filtration (Sepharose 4B) was developed to purify myosin. This procedure yielded myosin that was greater than 95% pure as judged by electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit composition of purified brain myosin was monitored by sodium dodecyl sulfate-polyacrylamide gel also containing a urea gradient. A closely migrating triplet in the heavy chain and three light chains, LC1, LC2, and LC3, of Mr 21,000, 19,000, and 17,000, respectively, were observed. These findings raise the possibility of the existence of myosin isoenzymes in the brain. Brain myosin formed bipolar thick filaments in 0.075 M KCl and MgCl2. At low ionic strength, the Mg2+-ATPase activity of myosin was stimulated 3- to 3.5-fold in the presence of skeletal muscle f-actin. Brain myosin also hydrolyzed other nucleotides; the rate of hydrolysis was ITP greater than ATP approximately equal to CTP greater than GTP approximately equal to UTP. The substrate (ATP) saturation curve in the presence of 10 mM CaCl2 and 0.6 M KCl was complex and consisted of plateau regions. The Arrhenius plot of the Ca-ATPase data was linear, whereas with ITPase, it was biphasic with a break occurring around 20 degrees C.

Plant pyruvate dehydrogenase complex purification, characterization and regulation by metabolites and phosphorylation.

The pyruvate dehydrogenase complex was purified from mitochondria of cauliflower, Brassica oleracea var. botrytis floral buds to a specific activity of 5.4 mumol of NADH/min per mg of protein. The pyruvate dehydrogenase complex required CoASH, NAD+, thiamine pyrophosphate and Mg2+ for the oxidative decarboxylation of pyruvate. The kinetic analysis of the complex gave a series of parallel lines for all substrates. Product interaction patterns showed that NADH is competitive with NAD+; acetyl-CoA is competitive with CoASH; and NADH and acetyl-CoA uncompetitive with pyruvate. These kinetic patterns suggest a multisite ping-pong mechanism as described by Cleveland ((1973) J. Biol. Chem 248, 8353). The noncompetitive inhibition of NADH versus CoASH, and acetyl-CoASH versus NAD are not predicted by this mechanism. Regulation of the complex was more sensitive to the NADH/NAD+ ratio than acetyl-CoA/CoASH ratio. Hydroxypyruvate and glyoxylate inhibited the complex noncompetitively versus pyruvate. The pyruvate dehydrogenase complex was inactivated and phosphorylated by ATP. The ATP dependent inactivation is believed to be enzyme catalyzed by a pyruvate dehydrogenase complex kinase. However, no evidence was found for a plant pyruvate dehydrogenase complex phosphatase. The results suggest that the cauliflower pyruvate dehydrogenase complex is regulated by a phosphorylation-dephosphorylation mechanism.