Properties of a calcium-activated protease in squid axoplasm which selectively degrades neurofilament proteins

Axoplasm extruded from the giant axon of the squid contains Ca²⁺-activated proteases. The protease in the 100,000X g of supernatant of axoplasm is very specific and degrades only the 200,000 MW, neurofilament protein (NF₂₀₀), whereas the protease(s) in the pellet has a much wider range of substrate specificity. The activation of the supernatant protease is restricted to the Ca²⁺ ion, and no other divalent cation will substitute. The protease requires Ca²⁺ at a higher concentration than 0.5 mM for activation, and has a pH optimum of about 7.5. Degradation of the NF₂₀₀ appears to proceed through a 100,000 MW and possibly a 47,000–50,000-MW intermediate form before degradation to TCA-soluble peptides. Activity of the protease is inhibited by divalent cation chelators, Cu²⁺ and Fe²⁺, sulphydryl inhibitors, and leupeptin. This specific Ca²⁺-activated protease in squid axoplasm has identical properties to Ca²⁺-activated proteases found in various non-neural tissues. Despite its narrow protein substrate specificity, Ca²⁺-activated protease purified from human platelets effectively degrades squid NF₂₀₀, suggesting a possible structural relationship between platelet and muscle actin-binding proteins and neurofilament proteins.     

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.     

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.     

Magnesium influx in dialyzed squid axons

Summary The influx of magnesium from seawater into squid giant axons has been measured under conditions where internal solute control in the axon was maintained by dialysis. Mg influx is smallest (1 pmol/cm² sec) when both Na and ATP have been removed from the axoplasm by dialysis. The addition of 3mm ATP to the dialysis fluid gives a Mg influx of 2.5 pmol/cm² sec while the addition of [Na] _i and [ATP] _i gives 3 pmol/cm² sec as a value for Mg influx; this corresponds well with fluxes measured in intact squid giant axons.The Mg content of squid axons is 6 mmol/kg axoplasm; this is unaffected by soaking axons in Li or Na seawater for periods of up to 100 min.     

CONFIGURATION OF A FILAMENTOUS NETWORK IN THE AXOPLASM OF THE SQUID (LOLIGO PEALII L.) GIANT NERVE FIBER

High-resolution electron microscopy is integrated with physicochemical methods in order to investigate the following preparations of the giant nerve fibers of the squid (Loligo pealii L.): (1) Thin sections of fibers fixed in four different fixatives; (2) fresh axoplasm stained negatively in solutions of different pH and composition; (3) chemically isolated threadlike elements of the axoplasm. A continuous, three-dimensional network can be identified in all these preparations of the axoplasm. The network is composed of coiled or looped unit-filaments ~30 A wide. The unit-filaments are intercoiled in strands ~ 70–250 A wide. The strands are oriented longitudinally in the axoplasm, often having a sinuous course and cross-associations. Microtubules are surrounded by intercoiled unit-filaments and filamentous strands. Calcium ions cause loosening and disintegration of the network configuration. UO₂⁺⁺ ions of a 1% uranyl acetate solution at pH 4.4 display a specific affinity for filamentous protein structures of the squid giant nerve fiber axoplasm, segregating the filamentous elements of the axoplasm in a coiled, threadlike preparation. The uranyl ions combine probably with the carboxyl groups of the main amino acids of the protein—glutamic and aspartic acids. It is proposed that by coiling/decoiling and folding/unfolding of the unit-filaments, shifts in physicochemical properties of the axoplasm are maintained.     

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.     

INCORPORATION OF LABELLED PHOSPHATE INTO PHOSPHOLIPIDS IN SQUID GIANT AXONS

Inorganic phosphate labelled with ³²P was applied to giant axons excised from squid (Loligo pealeii) by addition of ³²Pi to the bathing solution, by injection into the axon, or by addition to axoplasm which had been separated from the sheath. The preparations were kept at 10 to 25° for various times up to 4 hr. When ³²P_i was supplied by way of the bathing solution, axoplasm and sheath were usually separated at the end of incubation before extraction of the lipids. Lipids were extracted with chloroform-methanol and resolved by paper chromatography. The lipids which became labelled appeared to be the same in sheath and axoplasm. They were identified by cochromatography with known lipids and by chromatography of products formed from them by mild alkaline hydrolysis. They included phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid, and probably somelysophosphatidylethanolamine. Some labelled components remained unidentified. Phosphatidylcholine was apparently present, but did not become significantly labelled either in sheath or in axoplasm, or in a squid's stellate ganglion. There was no evidence that separation from the sheath impaired the capacity of the axoplasm for lipid synthesis.     

The presence of transfer RNA in the axoplasm of the squid giant axon

Previous work has revealed that 4S RNA is the primary species of RNA in the axoplasm from the giant axons of the squid and Myxicola. This study shows that axoplasmic 4S RNA from the squid giant axon has the functional properties of tRNA. Axoplasmic RNA was charged with amino acids by aminoacyl-tRNA synthetases prepared from squid brain. The aminoacylation was prevented by incubating the RNA with RNase prior to running the reaction. The amino acid-RNA complex was labile at pH 9, which is characteristic of the acyl linkage between an amino acid and its tRNA. Aminoacyl-tRNA synthetase activity was also present in the axoplasm, primarily in the soluble fraction.     

Effects of narcotics on the giant axon of the squid

—Levorphanol (10^-3 M) reversibly blocked conduction in the giant axon of the squid and axons from the walking legs of spider crab and lobster. Similar concentrations of levallorphan and dextrorphan blocked conduction in the squid giant axon. Under the same experimental condition morphine caused an approximately 40 per cent decrease in spike height. Levorphanol did not affect the resting potential or resistance of the squid axon. Spermidine, spermine and dinitrophenol had little or no direct effect on the action potential nor did they alter the potency of levorphanol. Concentrations of levorphanol as low as 5 × 10^-5 M blocked repetitive or spontaneous activity in the squid axon induced by decreasing the divalent cations in the medium. After exposure to tritiated levorphanol, the axoplasm and envelope of the squid axon accumulated up to 500 per cent of the concentration of tritium found in the external medium, dependent on time of exposure, and other variables. At pH 6 the levels of penetration were 33-50% of those found at pH 8, which correlates with our observation that levorphanol is about 33 % as potent in blocking the action potential at pH 6. The penetrability of levorphanol was not affected by spermidine, dinitrophenol or cottonmouth moccasin venom. Levorphanol did not alter the penetration of [C¹⁴]acetylcholine nor did it render the squid axon sensitive to it. The block of axonal conduction by compounds of the morphine series is discussed both as to possible mechanisms and significance.     

Regulation of intracellular calcium in squid axons

Internal dialysis and metallochromic indicators were used to determine the free calcium concentration and calcium buffering properties of squid axoplasm. The free calcium concentration in fresh unloaded squid axons is about 30 to 50 nM. About 6% of the calcium content (ca. 50 mumol/kg axoplasm) of a fresh squid axon is held in a metabolically labile, presumably mitochondrial, component. A morphological consequence of this finding is that there should be no accumulation of calcium in mitochondria of fresh squid axons unless there is a large component of nonlabile calcium. The physiological implication is that the mitochondria are probably not buffers for physiological perturbations in free calcium concentration. When an exogenous load of several hundred mumol/kg axoplasm with an ambient ionized calcium concentration above a few hundred nanomolar is applied to axoplasm, all of it goes into organelles. About one-third of that load is found in the mitochondria and about two thirds in some other organelles. When axoplasm is poisoned with carbonyl cyanide-p-trifluoromethonyphenylhydrazone (FCCP), around 70% of the load remains in the nonmitochondrial fraction.     

In situ accumulation of calcium by organelles of squid axoplasm

Existing morphological and physiological evidence indicates that axoplasm of squid axons sequesters calcium by both mitochondrial and non-mitochondrial buffers. The present work demonstrates that essentially all of the non-mitochondrial component is located in organelles. Extruded axoplasm was loaded with varying amounts of calcium by mixing with small volumes of solutions containing pH buffered ⁴⁵Ca. Ethyleneglycol-bis(β-amino-ethyl ether)N,N′-tetraacetic acid (EGTA) or diethylenetriamine pentaacetic acid (DTPA) was used to stabilize the free calcium. The axoplasm was then sucked up in a polyethylene tube and centrifuged at 100,000 g for 2–3 hours to produce a loose pellet comprising 10–20% of the axoplasm volume. After centrifugation, the tube was frozen, sliced into segments, and counted by liquid scintillation. No significant pellet accumulation of exogenous calcium occurred at physiological concentrations of free calcium (ca. 50 nM); however, a threshold for accumulation existed at 150–200 nM. Essentially complete pellet sequestration of the exogenous load occurred at a free calcium concentration above 1 μM. About half of the pellet buffering capacity was sensitive to carbonyl cyanide, p-trifluoromethoxy phenylhydrazone (FCCP). Variation of exogenous load between 0.1 – 3 mmole/kg axoplasm did not affect the buffering capacity of either the FCCP sensitive or insensitive components when the free calcium concentration was above threshold.     

Subcellular Localization of Functionally Differentiated Microtubules in Squid Neurons: Regional Distribution of Microtubule-Associated Proteins and β-Tubulin Isotypes

The subcellular localization of microtubule proteins in the neurons of squid (Doryteuthis bleekeri) was immunologically studied using monoclonal antibodies against the microtubule proteins. We found that (1) the squid neurons contained three kinds of high-molecular-weight microtubule-associated proteins [MAP A of approximately 300 kilodaltons (kD), MAP B of 260 kD, and axolinin of 260 kD] and two kinds of beta-tubulin isotypes (beta 1 and beta 2); (2) the cell body of the squid giant neuron contained MAP A, MAP B, and the two beta-tubulin isotypes (beta 1 and beta 2); (3) axolinin and the beta 1 isotype were present exclusively in the peripheral axoplasm of the giant axon; and (4) a small amount of axolinin, MAP A, and the beta 1 isotype was found in the insoluble aspect of the central axoplasm, whereas the soluble aspect of the central axoplasm contained an abundant amount of MAP A along with the modified form of the beta 1 isotype. The regional difference of the distribution of the microtubule protein components may explain the differences in stability among axonal microtubules. Microtubules in the soluble aspect of the central axoplasm are sensitive to any treatment with colchicine, cold temperature, and high ionic strength but those both in the insoluble aspect of the central axoplasm and in the peripheral axoplasm are highly insensitive to the treatment.     

Axoplasmic RNA species synthesized in the isolated squid giant axon

Isolated squid stellate nerves and giant fiber lobes were incubated for 8 hr in Millipore filtered sea water containing [³H]uridine. The electrophoretic patterns of radioactive RNA purified from the axoplasm of the giant axon and from the giant fiber lobe (cell bodies of the giant axon) demonstrated the presence of RNA species with mobilities corresponding to tRNA and rRNA. The presence of labeled rRNAs was confirmed by the behavior of the large rRNA component (31S) which, in the squid, readily dissociates into its two constituent moyeties (17S and 20S). Comparable results were obtained with the axonal sheath and the stellate nerve. In all the electrophoretic patterns, additional species of radioactive RNA migrated between the 4S and the 20S markers, i.e. with mobilities corresponding to presumptive mRNAs. Chromatographic analysis of the purified RNAs on oligo(dT)cellulose indicated the presence of labeled poly(A)⁺ RNA in all tissue samples. Radioactive poly(A)⁺ RNA represented approximately 1% of the total labeled RNA in the axoplasm, axonal sheath and stellate nerve, but more than 2% in the giant fiber lobe. The labeled poly(A)⁺ RNAs of the giant fibre lobe showed a prevalence of larger species in comparison to the axonal sheath and stellate nerve. In conclusion, the axoplasmic RNAs synthesized by the isolated squid giant axon appear to include all the major classes of axoplasmic RNAs, that is rRNA, tRNA and mRNA.Special Issue dedicated to Prof. Holger Hydén.     

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.     

Mg2+- or Ca2+-Activated ATPase in Squid Giant Fiber Axoplasm

A divalent cation-activated ATPase in axoplasm from the squid giant axon is described. The enzyme requires Mg2+ or Ca2+, has a K+ optimum of 60 mM, and has a pH optimum of 7.5. Several nucleotide triphosphates other than ATP can serve as substrates. The enzyme is inhibited by excess ATP or Mg2+. The enzyme is enriched in a rapidly sedimenting fraction of the axoplasm, and is eluted in the exclusion volume of a Sepharose 4B column, suggesting that it is associated with a highly aggregated structure. Comparison of the properties of enzyme with those of myosin and Na+-K+-ATPase suggests that differs from both of these enzymes. The enzyme has many similarities to vertebrate nerve ATPases previously described. The demonstration of the presence of this ATPase in squid axoplasm proves the neuronal localization of the enzyme.     

Phospholipid synthesis in the squid giant axon: incorporation of lipid precursors

The squid giant axon and extruded axoplasm from the giant axon were used to study the capacity of axoplasm for phospholipid synthesis. Extruded axoplasm, suspended in chemically defined media, catalyzed the synthesis of phospholipids from all of the precursors tested. 32P-Labeled inorganic phosphate and gamma-labeled ATP were actively incorporated into phosphatidylinositol phosphate, while [2-3H]myo-inositol and L-[3H(G)]serine were actively incorporated into phosphatidylinositol and phosphatidylserine, respectively. Though less well utilized. [2-3H]glycerol was incorporated into phosphatidic acid, phosphatidylinositol, and triglyceride, and methyl-3H]choline and [1-3H]ethanolamine were incorporated into phosphatidylcholine and phosphatidylethanolamine, respectively. Isolated squid giant axons were incubated in artificial seawater containing the above precursors. The axoplasm was extruded following the incubations. Although most of the product lipids were recovered in the sheath (composed of cortical axoplasm, axolemma, and surrounding satellite cells), significant amounts (4-20%) were present in the extruded axoplasm. With tritiated choline and myo-inositol, the major labeled phospholipids found in both the extruded axoplasm and the sheath were phosphatidylcholine and phosphatidylinositol, respectively. With both glycerol and phosphate, phosphatidylethanolamine was a major labeled lipid in both axoplasm and sheath. These findings demonstrate that all classes of phospholipids are formed by endogenous synthetic enzymes in axoplasm. In addition, we feel that the different patterns of incorporation by intact axons and extruded axoplasm indicate that surrounding sheath cells contribute lipids to axoplasm. A comprehensive picture of axonal lipid metabolism should include axoplasmic synthesis and glial-axon transfer as pathways complementing the axonal transport of perikaryally formed lipids.     

Alteration of birefringence signals from squid giant axons by intracellular perfusion with protease solution

The optical signal, arising from a transient birefringence change associated with excitation, was recorded from a squid giant axon together with the membrane potential change, and the effect of removal of the axoplasm on the optical signal was examined. In an unperfused axon, repetitive stimulation at a frequency of about 100 Hz produced two kinds of optical response. The initial response had a brief, spike-like time course and was elicited by each stimulating pulse. The delayed response had a slow time course and the sign of decreased light intensity, and summated with repetitive stimulation. Most of the axoplasm was removed from interior of the axon by intracellular perfusion with solutions containing pronase at a concentration of 0.1 mg/ml. The delayed response could selectively be eliminated by perfusion with a pronase-containing solution for 2–8 min. The result was interpreted as showing that the delayed birefringence signal originates from axoplasm when its gel structure was transiently disturbed by an increased Ca²⁺ influx associated with excitation. When perfusion was further continued the duration of the action potential started increasing and often a prominent after-depolarization appeared. At this stage the initial optical response was again followed by a large show signal with the sign of increased light intensity. This reversed delayed response was tentatively assumed to originate from the membrane with some remaining axoplasm, but its cause is still not understood.     

The distribution of nucleic acids in the giant axon of the squid (Loligo pealii)

Abstract— RNA and DNA were determined in the axoplasm and sheath of squid giant axons. If the RNA was related to axonal length, equivalent amounts of RNA were found in the axoplasm and sheath. However, when it was expressed as a function of dry wt. or residue protein, the concentration of RNA in the sheath was four times greater than in the axoplasm. A ratio of 1·1 was found for sheath RNA/sheath DNA. Less DNA was present in the axoplasm than the amount which could be accurately determined with the methods employed.     

Incorporation of H3-uridine and the isolation and characterization of RNA from squid axon

H₃-Uridine microinjected in the giant axons of the squid is incorporated in a TCA insoluble material. There is no difference between stimulated and resting axons as to the amount incorporated. The amount incorporated is increased if the stimulation precedes the microinjection of the tracer. RNA was purified and characterized from the axoplasm, axon sheaths and from a purified membrane preparation obtained from squid retinal nerve.     

The incorporation of microinjected 14C-amino acids into TCA insoluble fractions of the giant axon of the squid

Giant squid axons were microinjected with serine, valine and leucine-C¹⁴ under controlled electrophysiological conditions. These amino acids are incorporated into TCA insoluble fraction in the isolated axon. This incorporation is higher in the stimulated axons as compared to non-stimulated ones. By processing separately the axoplasm and axon sheath, it was found that the last one is responsible almost entirely for the observed incorporation. Through differential centrifugation of homogenates of microinjected axons was shown that the highest incorporation occurred in the 1500 × g sediment, which probably corresponds to membranes. The incorporation of amino acids in stimulated axons, is strongly inhibited by chloramphenicol and actinomycin D.