A CALCIUM ACTIVATED PROTEASE IN SQUID AXOPLASM

Evidence for a protease in squid axoplasm which is selectively activated by Ca²⁺ and blocked by SH-inhibitors is presented. This protease appears to be particularly effective in degrading squid neurofilament proteins, but also extensively degrades various other major protein components in axoplasm.     

Calcium-dependent proteins in platelets response of calcium-activated protease in normal and thrombasthenic platelets to aggregating agents

Recent studies have suggested a role for Ca²⁺-dependent proteolysis in the regulation of microfilament disassembly by high molecular weight actin-binding protein. A Ca²⁺-activated protease similar to myofibrillar Ca²⁺-activated protease has been described in platelets. To explore the role of Ca²⁺-activated proteolysis of actin-binding protein in platelet function, we have examined the effects of platelet aggregating agents on platelet Ca²⁺-activated protease-like activity. The hydrolysis of actin-binding protein by Ca²⁺-activated protease was determined electrophoretically. The calcium ionophore, A23187, produced a dose-dependent stimulation of Ca²⁺-activated protease-like activity in the presence of exogenous calcium but had no effect in the absence of external calcium. Both normal and thrombasthenic platelets generated Ca²⁺-activated protease-like activity in response to A23187. Ionophore-induced stimulation of Ca²⁺-activated protease-like activity was not affected by prior incubation of platelets with 8-bromo cyclic GMP, 8-bromo cyclic AMP, prostaglandin E₁, prostaglandin I₂, indomethacin or tetracaine, but was inhibited by the sulfhydryl inhibitor N-ethylmaleimide. These results confirm the presence of Ca²⁺-activated protease in platelets and indicate that the source of calcium important in Ca²⁺-activated protease stimulation is in part extracellular. Other aggregating agents, thrombin, epinephrine, and ADP, were not accompanied by hydrolysis of actin-binding protein, indicating that the alteration in ionic calcium that occurs during aggregation by these other agents is insufficient to generate Ca²⁺-activated protease-like activity as measured by the present analytical technique.     

Calcium-activated proteolytic activity in rat liver mitochondria

Soluble extracts from sonicated rat liver mitochondria and rat liver cytosol were each chromatographed on DEAE-cellulose columns, and the fractions assayed for Ca²⁺-activated proteolytic activity using ¹⁴C-casein as a substrate. The mitochondrial preparations were shown to be free of cytosolic and microsomal contamination by the lack of alcohol dehydrogenase activity, a cytosolic marker enzyme, and by a lack of cytochrome P-450 activity, a microsomal marker enzyme. Two peaks of Ca²⁺-activated neutral endoprotease activity were resolved from the mitochondrial fractions. One protease was half-maximally activated with 25 μM Ca²⁺, and the other by 750 μM Ca²⁺. Rat liver cytosol contained only a high Ca²⁺-requiring protease peak. This is the first demonstration of Ca²⁺-activated proteases in mitochondria.     

Characterization of phospholipase A2 and acyltransferase activities in squid (loligo pealei) axoplasm: comparison with enzyme activities in other neural tissues, axolemma and axoplasmic subfractions

Phospholipase A₂ and acyltransferase were assayed and characterized in pure axoplasm and neural tissues of squid. Intracellular phospholipase A₂ activity was highest in giant fiber lobe and axoplasm, followed by homogenates from retinal fibers, optic lobe and fin nerve. In most preparations, exogenous calcium (5 mM) caused a slight stimulation of activity. EGTA (2 mM) was somewhat inhibitory, indicating that low levels of endogenous calcium may be required for optimum activity. Phospholipase A₂ was inhibited by 0.1 mM p-bromophenacylbromide, and was completely inactivated following heating.The level of acylCoA: lysophosphatidylcholine acyltransferase activity was higher in axoplasm and giant fiber lobe than in other neural tissues of the squid. K_m (apparent) and V_max (apparent) for oleoyl-CoA and lysophosphatidylcholine were quite similar for axoplasm and giant fiber lobe enzyme preparations. Acyltransferase activity was inactivated by heat treatment, and greatly inhibited by 0.2 mM p-chloromercuribenzoate, and to a lesser extent by 20 mM N-ethylmaleimide.Phospholipase A₂ activity was present in fractions enriched in axolemmal membranes (separated from squid retinal fibers and garfish olfactory nerve) from both tissues, and it was also highly concentrated in vesicles derived from squid axoplasm. In all three preparations, phospholipase A₂ activity was stimulated by Ca⁺⁺ (5 mM) and inhibited by EGTA (2 mM). In addition, axoplasmic cytosol (114,000 g supernatant) retained a substantial portion of a Ca⁺⁺-independent phospholipase A₂, active in the presence of 2 mM EGTA. Acyltransferase activity was present at high content in both axolemma membrane rich fractions, and among subaxoplasmic fractions and axoplasmic vesicles.     

Identification of Ca2+-Activated Neutral Protease in the Peripheral Nerve and Its Effects on Neurofilament Degeneration

Rat sciatic nerve segments were incubated in five different media. Disappearance of neurofilament (NF) triplet proteins (200K, 160K, and 68K MW) occurred in medium containing Ca²⁺ and was inhibited by the addition of E-64-c or leupeptin. Therefore, the presence in the peripheral nerve of an enzyme whose properties are similar to those of Ca²⁺-activated neutral protease (CANP) is suggested. The extraction of crude CANP from rat sciatic nerve was performed. CANP activity was completely recovered (0.129 ± 0.008 U/g) in the precipitate salted out by the addition of 0 to 50% saturated ammonium sulfate to the soluble fraction of the peripheral nerve (crude CANP). Properties of the crude CANP were examined using NF as a substrate and were found to be similar to those of the CANP extracted from skeletal muscle. Identification of the crude CANP with the CANP extracted from rat skeletal muscle was performed using the immunoreplica method. Bands corresponding to 73K were detected in both CANPs.     

Evidence for the utilization of extracellular [γ-32P]ATP for the phosphorylation of intracellular proteins in the squid giant axon

Proteins in the squid giant axon were labeled with ³²P by in vitro incubation of isolated axoplasm with radioactive [γ-³²P]adenosine triphosphate (ATP) and separated by polyacrylamide sodium dodecyl sulfate gel electrophoresis. The two major phosphorylated regions on the gel had molecular weights of 400 000 and 200 000. These two peaks appear to be neurofilament proteins of squid axoplasm. The same set of proteins was phosphorylated in the axoplasm regardless of whether the [γ-³²P]ATP was applied in situ intracellularly or extracelarly. These results suggest that ATP in the extracellular space is, by some ATP-translocation mechanism, utilized in the process of intracellular phosphorylation. Measurements of the apparent influx of ATP across the squid axon membrane yielded results consistent with the view that ATP in the extracellular fluid could be transported into the axoplasm.     

Metabolic studies in vitro of the CNS cytoskeletal proteins: Synthesis and degradation

General aspects of metabolic features of the most prominent CNS intermediate filament proteins, the 200,000 (200K), 150,000 (150K), and 70,000 (70K) dalton proteins of the neuron, and the glial fibrillary acidic protein (GFAP) have been explored using the incubated spinal cord slice from the rat. Measurement of shortterm uptake of³H-labeled amino acids into the individual proteins separated on polyacrylamide gels revealed that of the three neurofilament proteins, 200K was most metabolically active, 150K was less active, and 70K contained very little incorporated radioactivity. Glial fibrillary acidic protein based on Coomassie blue stain affinity showed less metabolic activity than any of the neurofilament proteins. Those relationships were constant at all ages, but the metabolic activity of all CNS intermediate filaments decreased with age. When Ca²⁺ was present in the medium of the incubated slices, the intermediate filaments were rapidly destroyed, but GFAP was more resistant to degradation than the neurofilament proteins. GFAP and probably the neurofilament proteins also were relatively resistant to Ca²⁺-activated degradative mechanisms in spinal cords of rats at younger ages (15 day) than in those of older animals (10–18 months). It is likely that the Ca²⁺ activated protease is less active in developing animals in which the nerve tracts are still elongating, than in adults. These results suggest that GFAP is less active metabolically and more resistant to degradation than the neurofilament proteins at all stages of maturation, but that metabolic activity of all CNS intermediate filaments decreases with age while the susceptibility to degradation increases.Special Issue dedicated to Dr. Elizabeth Roboz-Einstein.     

Divalent Cations Modulate TMEM16A Calcium-Activated Chloride Channels by a Common Mechanism

The gating of Ca²⁺-activated Cl^? channels is controlled by a complex interplay among [Ca²⁺]_i, membrane potential and permeant anions. Besides Ca²⁺, Ba²⁺ also can activate both TMEM16A and TMEM16B. This study reports the effects of several divalent cations as regulators of TMEM16A channels stably expressed in HEK293T cells. Among the divalent cations that activate TMEM16A, Ca²⁺ is most effective, followed by Sr²⁺ and Ni²⁺, which have similar affinity, while Mg²⁺ is ineffective. Zn²⁺ does not activate TMEM16A but inhibits the Ca²⁺-activated chloride currents. Maximally effective concentrations of Sr²⁺ and Ni²⁺ occluded activation of the TMEM16A current by Ca²⁺, which suggests that Ca²⁺, Sr²⁺ and Ni²⁺ all regulate the channel by the same mechanism.     

Elevated levels of a calcium-activated muscle protease in rapidly atrophying muscles from vitamin E-deficient rabbits

A Ca²⁺-activated proteolytic enzyme ¹ that partially degrades myofibrials was isolated from hind limb muscles of normal rabbits and rabbits undergoing rapid muscle atrophy as a result of vitamin E deficiency. Extractable Ca²⁺-activated protease activity was 3.6 times higher in muscle tissue from vitamin E-deficient rabbits than from muscle tissue of control rabbits. Ultrastructural studies of muscle from vitamin E-deficient rabbits showed that the Z disk was the first myofibrillar structure to show degradative changes in atrophying muscle. Myofibris prepared from muscles vitamin E-deficient rabbits showed partial or complete loss of Z-disk density. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the amount of troponin-T (37 000 daltons) and α-actinin (96 000 daltons) was reduced in myofibrils from atrophying muscle as compared to myofibrils prepared from control muscle. In vitro treatment of purified myofibrils with purified Ca²⁺-activated proteolytic enzyme produced alterations in myofibrillar ultrastructure that were identical to the initial alterations occuring in myofibrils from atrophying muscle (i.e. weakening and subsequent removal of Z disks). Additionally the electrophoretic banding pattern of Ca²⁺-activated proteolytic enzyme-treated myofibrils is very similar to that of myofibrils prepared from muscles atrophying as a result of nutritional vitamin E deficiency. The possible role of Ca²⁺-activated proteolytic enzyme in disassembly and degradation of the myofibril is discussed.     

Efficient degradationin vitro of all intermediate filament subunit proteins by the Ca2+-activated neutral thiol proteinase from Ehrlich ascites tumor cells and porcine kidney

Vimentin, desmin, glial fibrillary acidic protein, neurofilament triplet proteins, and a mixture of cytokeratins were digested with Ca²⁺-activated neutral thiol proteinase isolated from Ehrlich ascites tumor (EAT) cells and porcine kidney. All intermediate filament proteins were degraded by the proteinase, although with different rates and Ca²⁺ optima. These results are in part at variance with our previous statement that the Ca²⁺-activated proteinase from EAT cells is specific for vimentin and desmin.     

Degradation of purified neurofilament subunits by calcium-activated neutral protease: characterization of the cleavage products

Neurofilament proteins (NFP) purified from rat spinal cord were labeled with ¹²⁵-I and incubated with a crude extract from rat spinal cord containing Ca²⁺-activated protease(s). The protease(s) activated by mM Ca²⁺ cleaved the NFP and produced a series of breakdown products which were different for each NFP. The amount of cleavage was dependent upon the incubation time with proteases but the pattern remained constant. Some of the cleavage products were relatively stable. These observations suggest that the cleavage products produced by treating NFP subunits with the endogenous protease can be used as a finger print to further study NFP metabolism and to better understand their role in physiological and pathological conditions of the nervous system.     

PROTEIN DEGRADATION IN SQUID GIANT AXONS

Axoplasm extruded from giant axons of the Chilean squid, Dosidicus gigas, contained a low level of neutral proteinase-like activity, equivalent to 4 × 10^?6 mg of chymotrypsin per mg of axoplasmic protein. The enzyme was active at physiological pH and ionic strength. Activity was completely inhibited by 1 mM-para-hydroxymercuribenzoate and was enhanced by divalent metal cations, especially Ca²⁺. Axoplasm also exhibited proteinase activity at pH 4.8. Both neutral and acid proteinase like activities were also present in the axonal sheath containing Schwann cells, but their specific activities relative to those in the axoplasm were different. A physiological role, related to the axoplasmic flow of protein, is discussed for the axoplasmic neutral proteinase-like activity.     

Activation of Cl− channels by extracellular Ca2+ in freshly isolated rabbit osteoclasts

Ionic channels regulated by extracellular Ca²⁺ concentration ([Ca²⁺]₀) were examined in freshly isolated rabbit osteoclasts. K⁺ current was suppressed by intracellular and extracellular Cs⁺ ions. In this condition, high [Ca²⁺]₀ evoked an outwardly rectifying current with a reversal potential of about −25 mV. When the concentration of extracellular Cl⁻ ions was altered, the reversal potential of the outwardly rectifying current shifted as predicted by the Nernst equation. 4′,4-diisothiocyanostilbene-2′,2-disulphonic acid (DIDS) inhibited the outwardly rectifying current. These results indicated that this current was carried through Cl⁻ channels. Cd²⁺ or Ni²⁺ caused a transient activation of the Cl⁻ current in contrast to the sustained activation elicited by Ca²⁺. Intracellular 20 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) inhibited the divalent cation-induced Cl⁻ current. Either when the osmolarity of extracellular medium was increased, or when 100 μM cAMP was dissolved in the patch pipette solution, high [Ca²⁺]₀ still elicited the Cl⁻ current, indicating that the divalent cation-induced Cl⁻ current was carried through Ca²⁺-activated Cl⁻ channels. Under perforated whole cell clamp extracellular divalent cations evoked the Cl⁻ current, indicating that the activation of Cl⁻ current did not arise from possible leakage of divalent cations from the extracellular medium under the whole cell clamp condition. This experiment further excluded a possible activation of volume-sensitive Cl⁻ channels under whole cell clamp. Intracellular application of guanosine 5′-O-(3-thiotriphosphate) (GTPγS) activated the Cl⁻ current and it was inhibited by intracellular 20 mM EGTA, suggesting that the activation of Cl⁻ current was mediated through a G protein, and that an increase in [Ca²⁺]_i was critical for the activation of Cl⁻ channels. A protein phosphatase inhibitor, okadaic acid (100 nM), caused an irreversible activation of the Cl⁻ current, suggesting that protein phosphatase 1 or 2A was involved in the regulation of Ca²⁺-activated Cl⁻ channels. © 1996 Wiley-Liss, Inc.     

Identification and purification of a soluble adenosine triphosphatase from Tetrahymena pyriformis .

An unusual ATPase isolated from the postribosomal supernatant fraction of $\text{Tetrahymena pyriformis}$ has been purified to homogeneity. The purification procedure consisted of protamine sulfate and heat treatment; column chromatography successively on phosphocellulose, DEAE-cellulose and Sephadex G-150; and isoelectric focusing. The pure enzyme has a molecular weight of 89,000 and requires either Ca²⁺ or Ba²⁺ for maximum activation. Nucleoside triphosphates are hydrolized at decreasing rates in the order: ATP > GTP > ITP > CTP > UTP. The K_m for ATP is 2.5 mM. Because of its properties the enzyme is tentatively classified as a soluble Ca²⁺-activated ATPase.     

Identification, Characterization and Partial Purification of a Thiol-protease Which Cleaves Specifically the Skeletal Muscle Ryanodine Receptor/Ca2+ Release Channel

A 94 kDa large subunit thiol-protease, as identified by anti-calpain antibodies, has been isolated from skeletal muscle junctional sarcoplasmic reticulum (SR). This protease cleaves specifically the skeletal muscle ryanodine receptor (RyR)/Ca²⁺ release channel at one site resulting in the 375 kDa and 150 kDa fragments. The 94 kDa thiol-protease degrades neither other SR proteins nor the ryanodine receptor of cardiac nor brain membranes. The partially purified 94 kDa protease, like the SR associated protease, had an optimal pH of about 7.0, was absolutely dependent on the presence of thiol reducing reagents, and was completely inhibited by HgCl₂, leupeptin and the specific calpain I inhibitor. However, while the SR membrane-associated protease requires Ca²⁺ at a submicromolar concentration, the isolated thiol-protease has lost the Ca²⁺ requirement. The 94 kDa thiol-protease had no effect on ryanodine binding but modified the channel activity of RyR reconstituted into planar lipid bilayer: in a time-dependent manner, the channel activity decreases and within several minutes the channel is converted into a subconducting state. The protease-modified channel activity is still Ca²⁺-dependent and ryanodine sensitive. This 94 kDa thiol-protease cross react with anti-calpain antibodies thus, may represent the novel large subunit of the skeletal muscle specific calpain p94. Received: 10 December 1996/Revised: 11 August 1997     

In vitro polymorphism and phase transitions of the neurofilamentous network isolated from the giant axon of the squid (Loligo pealei L.)

Summary Using electron microscopy (EM), optical diffraction and image reconstruction techniques, we have demonstrated polymorphism of neurofilamentous network (NFN) in vitro based on phase transitions of the protein assemblies. The specific polymorphic appearances depended upon a number of factors, such as K ⁺, Mg^{2 +}, Ca²⁺ ions, as well as the charge and hydration state of the molecules. Furthermore, modifications initiated by the state of phosphorylation of the sidearm proteins played an important role, especially in determining the sidearm disposition of the NFN. The Ca^{2 +}-activated protease removed the sidearms. Other enzymes activated by Ca^{2 +} may initiate new association patterns of the peptide remnants and the intercoiling of two smooth neurofilaments (NFs) into paired helical filament-like (PHF-like) strands. Prolonged storage of the isolated NFs in Rubinson-Baker solution resulted in autocrosslinking and intercoiling of modified NFN components. The in vitro polymorphism and phase transitions of squid NFN induced under controlled conditions have been compared to modifications of cytoskeleton observed by EM in frontal lobe biopsies of Alzheimer patients. We conclude that similar processes, as induced in vitro, do occur in neurons of Alzheimer patients.     

The spatial distribution of cations in nematocytes of Hydra vulgaris

The spatial distribution of cations was assayed qualitatively and quantitatively in tentacular nematocytes of Hydra vulgaris in a scanning transmission electron microscope by means of x-ray microanalysis performed on 100 nm thick freeze-dried cryosections. The matrix of undischarged cysts (stenoteles, desmonemes and isorhizas) was found to contain mainly K⁺. In isolated nematocysts of Hydra the intracapsular potassium can be readily substituted by practically any other mono- and divalent cation (Na⁺, NH₄ ⁺, Mn²⁺, Co²⁺, Mg²⁺, Ca²⁺, Fe²⁺) all, except Fe²⁺, without impairing the ability of the cyst to respond to the chemical triggering with dithioerythritol or proteases. Monovalent cations increase the osmotically generated intracapsular pressure compared to divalent ions.     

Purification and characterization of the cuticle-degrading protease produced by the entomopathogenic fungus,Beauveria bassiana in the presence of Sunn pest,Eurygaster integriceps (Hemiptera: Scutelleridae) cuticle

The extracellular protease from the entomopathogenic fungus, Beauveria bassiana in the presence of Eurygaster integriceps cuticle was isolated, purified and characterized. Isolate B1 of B. bassiana that shows high virulence against E. integriceps was examined for the production of the cuticle-degrading proteases. Results showed that both subtilisin-like (Pr1) and trypsin-like (Pr2) cuticle-degrading proteases were produced and the enzyme kinetic properties showed better activity of Pr1 in comparison with Pr2. The proteases were purified using acetone precipitation, Sephadex G-100 gel filtration and CM-Sepharose ion exchange chromatography, with a 5.09-fold increase in specific activity and 21.86% recovery. The enzyme molecular weight was estimated to be 47 kDa and the optimal pH and temperature were 8 and 45°C, respectively. The purified protease was activated by divalent cations, Ca^{2 +} and Mg^{2 +}, and inhibited by NaCl, KCl and determined as a serine protease by inhibition of its activity due to using PMSF, EDTA, mercaptoethanol and SDS. Studies on the timing of the protease secretion in the presence of cuticular substrates could provide information about the role of the accumulated hydrolytic enzymes during pathogenesis to better understand these processes.     

Distribution of Acid Protease Activity in the Squid Nervous System

Abstract: Acid protease activity was measured in homogenized stellate ganglion, axoplasm extruded from the squid giant axon, homogenized fin nerves, and in lysed synaptosomes prepared from the optic lobe of the squid. At least two different acid protease classes were distinguished on the bases of their inhibitor profiles. Acid protease activity was present in each of the above tissues except extruded axoplasm. This result suggests that the acid protease activity found in our homogenized finnerves might be located not within the axons but rather in glial cells or extracellular tissue. The absence of acid protease activity in extruded axoplasm indicates that acid proteases are unlikely to play a significant role in the catabolism of intracellular proteins along the length of the axon.     

Flash-induced consumption of molecular oxygen on the donor side of photosystem II in Mn-depleted subchloroplast membrane fragments: specific effects of manganese and calcium ions

It has been shown that removal of manganese from the water-oxidizing complex (WOC) of photosystem II (PSII) leads to flash-induced oxygen consumption (FIOC) which is activated by low concentration of Mn²⁺ (Yanykin et al., Biochim Biophys Acta 1797:516–523, 2010). In the present work, we examined the effect of transition and non-transition divalent metal ions on FIOC in Mn-depleted PSII (apo-WOC-PSII) preparations. It was shown that only Mn²⁺ ions are able to activate FIOC while other transition metal ions (Fe²⁺, V²⁺ and Cr²⁺) capable of electron donation to the apo-WOC-PSII suppressed the photoconsumption of O₂. Co²⁺ ions with a high redox potential (E ⁰ for Co²⁺/Co³⁺ is 1.8 V) showed no effect. Non-transition metal ions Ca²⁺ by Mg²⁺ did not stimulate FIOC. However, Ca²⁺ (in contrast to Mg²⁺) showed an additional activation effect in the presence of exogenic Mn²⁺. The Ca²⁺ effect depended on the concentration of both Mn²⁺ and Ca²⁺. The Ca effect was only observed when: (1) the activation of FIOC induced by Mn²⁺ did not reach its maximum, (2) the concentration of Ca²⁺ did not exceed 40 μM; at higher concentrations Ca²⁺ inhibited the Mn²⁺-activated O₂ photoconsumption. Replacement of Ca²⁺ by Mg²⁺ led to a suppression of Mn²⁺-activated O₂ photoconsumption; while, addition of Ca²⁺ resulted in elimination of the Mg²⁺ inhibitory effect and activation of FIOC. Thus, only Mn²⁺ and Ca²⁺ (which are constituents of the WOC) have specific effects of activation of FIOC in apo-WOC-PSII preparations. Possible reactions involving Mn²⁺ and Ca²⁺ which could lead to the activation of FIOC in the apo-WOC-PSII are discussed.