IDENTIFICATION OF A CALCIUM-BINDING, BRAIN SPECIFIC PROTEIN IN THE AXOPLASM OF SQUID GIANT AXONS

Abstract— An acidic protein has been isolated from the optic lobes of two cephalopods, Sepia officinalis and Loligo vulgaris. The protein has been obtained in pure form by fractionation with ammonium sulphate and chromatography on DEAE-cellulose and Sephadex G 100. Its apparent molecular weight is 13,000–15,000. Glutamic and aspartic acids account for 35 per cent of the amino acid residues. The protein binds Ca²⁺ ions with an apparent dissociation constant of 2·5 × 10⁻⁵ M at physiological concentrations of KCI. Antibodies have been prepared against the protein purified from Sepia officinalis. By the micro-complement fixation technique it has been shown that the protein is highly concentrated in the nervous system of cephalopods and that the amount in the axoplasm of squid giant axons is eight to nine-fold higher than in the optic lobes of the same animal.     

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.     

CALCIUM-CONTAINING ELECTRON-DENSE STRUCTURES IN THE AXONS OF THE SQUID GIANT SYNAPSE

Following the Oschman and Wall technique, electron-dense structures (EDS) were found on unstained, unosmicated membranes of squid giant synapse axons. These densities contain high concentrations of calcium and phosphorus as identified by energy dispersive X-ray analysis. Based on the signal strength, the quantity is significantly greater than that of other regions of the membrane or tissue spaces. The calcium EDS occur as plaques or globules along the axonic membrane, and small globules are found between sheath cell processes. EDS also occur at the synaptic site. These densities were correlated with the opacity change seen in giant axons. It is proposed that these structures represent sites where the calcium-binding protein found by other investigators has become nearly saturated with calcium.     

PHOSPHOLIPASE ACTIVITY IN SQUID AND FROG AXONS

Evidence for the presence of phosphatide acylhydrolase activity (EC 3.1.1) in centrifuged homogenate supernatants and extracts of squid giant axons and centrifuged homogenate supernatants of frog sciatic nerve bundles is reported. The enzyme was assayed by measurement of the rate of deacylation of [U-¹⁴C]phosphatidyl choline. The deacylation activity in the nerve homogenate supernatants exhibits: a pH maximum at 7.2–7.4 (25°C); a calcium ion maximum at 12–13 mM-CaCl₂(aq); a K_m value of 3.4 × 10^?4 M (25°C); and a temperature maximum at 37°C. The activation energy over the range 8–37°C is 5.7 ± 0.2kcal-mol^?1.     

RECTIFICATION AND INDUCTANCE IN THE SQUID GIANT AXON

Previous measurements have shown that the electrical properties of the squid axon membrane are approximately equivalent to those of a circuit containing a capacity shunted by an inductance and a rectifier in series. Selective ion permeability of a membrane separating two electrolytes may be expected to give rise to the rectification. A quasi-crystalline piezoelectric structure of the membrane is a plausible explanation of the inductance. Some approximate calculations of behavior of an axon with these membrane characteristics have been made. Fair agreement is obtained with the observed constant current subthreshold potential and impedance during the foot of the action potential. In a simple case a formal analogy is found between the calculated membrane potential and the excitability defined by the two factor formulations of excitation. Several excitation phenomena may then be explained semi-quantitatively by further assuming the excitability proportional to the membrane potential. Some previous measurements and subthreshold potential and excitability observations are not consistent with the circuit considered and indicate that this circuit is only approximately equivalent to the membrane.     

MEMBRANE AND PROTOPLASM RESISTANCE IN THE SQUID GIANT AXON

The direct current longitudinal resistance of the squid giant axon was measured as a function of the electrode separation. Large sea water electrodes were used and the inter-electrode length was immersed in oil. The slope of the resistance vs. separation curve is large for a small electrode separation, but becomes smaller and finally constant as the separation is increased. An analysis of the resistance vs. length curves gives the following results. The nerve membrane has a resistance of about 1000 ohm cm.² The protoplasm has a specific resistance of about 1.4 times that of sea water. The resistance of the connective tissue sheath outside the fiber corresponds to a layer of sea water about 20µ in thickness. The characteristic length for the axon is about 2.3 mm. in oil and 6.0 mm. in sea water.     

TRANSPORT OF MICROTUBULAR PROTEIN IN AXONS OF RETINAL GANGLION CELLS

Abstract— Following intraocular injection of [³H]leucine, microtubular protein was isolated by two different methods from the optic pathway of the rabbit. It was found that most of this protein was transported at a slow rate in the axons of retinal ganglion cells. After arrival at the nerve terminals in the lateral geniculate body, the microtubular protein decayed with a half-life of 9 days.     

LONGITUDINAL IMPEDANCE OF THE SQUID GIANT AXON

Longitudinal alternating current impedance measurements have been made on the squid giant axon over the frequency range from 30 cycles per second to 200 kc. per second. Large sea water electrodes were used and the inter-electrode length was immersed in oil. The impedance at high frequency was approximately as predicted theoretically on the basis of the poorly conducting dielectric characteristics of the membrane previously determined. For the large majority of the axons, the impedance reached a maximum at a low frequency and the reactance then vanished at a frequency between 150 and 300 cycles per second. Below this frequency, the reactance was inductive, reaching a maximum and then approaching zero as the frequency was decreased. The inductive reactance is a property of the axon and requires that it contain an inductive structure. The variation of the impedance with interpolar distance indicates that the inductance is in the membrane. The impedance characteristics of the membrane as calculated from the measured longitudinal impedance of the axon may be expressed by an equivalent membrane circuit containing inductance, capacity, and resistance. For a square centimeter of membrane the capacity of 1 µf with dielectric loss is shunted by the series combination of a resistance of 400 ohms and an inductance of one-fifth henry.     

THE SYNTHESIS OF CHOLINE PHOSPHOGLYCERIDES IN THE GIANT FIBRE SYSTEM OF THE SQUID

The mechanisms and pathways of synthesis of phosphatidylcholine in the giant fibre system of the squid (Loligo vulgaris) have been examined by incubating the stellate ganglion-nerve preparation or its separated compartments in an artificial bathing solution with labelled choline. Other experiments were done by dissecting the whole stellate ganglion into axoplasm, axon sheath, giant fibre lobe, small fibres and ganglion residue, after incubation. The initial rate of choline incorporation into choline phosphoglycerides was severalfold higher in the lobe than in the axon. Higher lipid radioactivity was recovered in the axon sheath as compared to the axoplasm, and in the small fibres as compared to the ganglion residue which contains its cell bodies. The production of phosphorylcholine and CDP-choline in the intact ganglion-nerve preparation during incubation with choline points to the occurrence of the net synthesis pathway for phosphatidylcholine in this material. Base-exchange activity was also observed in the axon and giant fibre lobe preparations in vitro, but no indication can yet be given whether it also takes place in intact preparations. Electrical stimulation and‘depolarizing’conditions enhance choline phosphorylation in the squid axon and lobe, but decrease phosphatidylcholine labelling.     

TRANSVERSE ELECTRIC IMPEDANCE OF THE SQUID GIANT AXON

The impedance of the excised giant axon from hindmost stellar nerve of Loligo pealii has been measured over the frequency range from 1 to 2500 kilocycles per second. The measurements have been made with the current flow perpendicular to the axis of the axon to permit a relatively simple analysis of the data. It has been found that the axon membrane has a polarization impedance with an average phase angle of 76° and an average capacity of 1.1µf./cm² at 1 kilocycle. The direct current resistance of the membrane could not be measured, but was greater than 3 ohm cm.² and the average internal specific resistance was four times that of sea water. There was no detectable change in the membrane impedance when the axon lost excitability, but some time later it decreased to zero.     

ELECTRIC IMPEDANCE OF THE SQUID GIANT AXON DURING ACTIVITY

Alternating current impedance measurements have been made over a wide frequency range on the giant axon from the stellar nerve of the squid, Loligo pealii, during the passage of a nerve impulse. The transverse impedance was measured between narrow electrodes on either side of the axon with a Wheatstone bridge having an amplifier and cathode ray oscillograph for detector. When the bridge was balanced, the resting axon gave a narrow line on the oscillograph screen as a sweep circuit moved the spot across. As an impulse passed between impedance electrodes after the axon had been stimulated at one end, the oscillograph line first broadened into a band, indicating a bridge unbalance, and then narrowed down to balance during recovery. From measurements made during the passage of the impulse and appropriate analysis, it was found that the membrane phase angle was unchanged, the membrane capacity decreased about 2 per cent, while the membrane conductance fell from a resting value of 1000 ohm cm.² to an average of 25 ohm cm.² The onset of the resistance change occurs somewhat after the start of the monophasic action potential, but coincides quite closely with the point of inflection on the rising phase, where the membrane current reverses in direction, corresponding to a decrease in the membrane electromotive force. This E.M.F. and the conductance are closely associated properties of the membrane, and their sudden changes constitute, or are due to, the activity which is responsible for the all-or-none law and the initiation and propagation of the nerve impulse. These results correspond to those previously found for Nitella and lead us to expect similar phenomena in other nerve fibers.     

SITE OF BIOSYNTHESIS OF BRAIN-SPECIFIC PROTEINS IN THE GIANT FIBRE SYSTEM OF THE SQUID

The synthesis of brain-specific proteins has been examined in perikaryal and axonal regions of the giant fibre system of the squid. After in vitro incubation of stellate ganglia, stellate nerves and isolated giant axons with radioactive amino acids, the labelled soluble proteins have been extracted from the giant fibre lobe, the axoplasm and the axonal sheath of the giant axon and have been separated by gel electrophoresis on a continuous system. In addition, they have been challenged with antisera prepared against the cephalopod brain-specific proteins L1 and L2 and the resulting precipitate has been resolved by sodium dodecyl sulphate-gel electrophoresis. Synthesis of these two proteins appears to be restricted to the giant fibre lobe, while an additional discrete protein band (L5) also becomes clearly labelled in the isolated giant axon. Radioactive components migrating in the region of the L1 and L2 proteins are synthesized in the isolated giant axon. They can be distinguished from tbese proteins on the basis of electrophoretic and immunochemical criteria.     

TRANSVERSE IMPEDANCE OF THE SQUID GIANT AXON DURING CURRENT FLOW

The change in the transverse impedance of the squid giant axon caused by direct current flow has been measured at frequencies from 1 kc. per second to 500 kc. per second. The impedance change is equivalent to an increase of membrane conductance at the cathode to a maximum value approximately the same as that obtained during activity and a decrease at the anode to a minimum not far from zero. There is no evidence of appreciable membrane capacity change in either case. It then follows that the membrane has the electrical characteristics of a rectifier. Interpreting the membrane conductance as a measure of ion permeability, this permeability is increased at the cathode and decreased at the anode.     

MEMBRANE POTENTIAL OF THE SQUID GIANT AXON DURING CURRENT FLOW

The squid giant axon was placed in a shallow narrow trough and current was sent in at two electrodes in opposite sides of the trough and out at a third electrode several centimeters away. The potential difference across the membrane was measured between an inside fine capillary electrode with its tip in the axoplasm between the pair of polarizing electrodes, and an outside capillary electrode with its tip flush with the surface of one polarizing electrode. The initial transient was roughly exponential at the anode make and damped oscillatory at the sub-threshold cathode make with the action potential arising from the first maximum when threshold was reached. The constant change of membrane potential, after the initial transient, was measured as a function of the total polarizing current and from these data the membrane potential is obtained as a function of the membrane current density. The absolute value of the resting membrane resistance approached at low polarizing currents is about 23 ohm cm.². This low value is considered to be a result of the puncture of the axon. The membrane was found to be an excellent rectifier with a ratio of about one hundred between the high resistance at the anode and the low resistance at the cathode for the current range investigated. On the assumption that the membrane conductance is a measure of its ion permeability, these experiments show an increase of ion permeability under a cathode and a decrease under an anode.     

ACETYLCHOLINE CONTENT OF THE SCHWANN CELL AND AXON IN THE GIANT NERVE FIBRE OF THE SQUID

Abstract— Acetylcholine and choline were identified and their concentrations measured, by means of gas chromatography/mass spectrometry, in extracts obtained from nerve fibers of the hindmost stellar nerve of the squid Sepioteuthis sepioidea. These compounds were quantitated in samples of stellar nerve devoid of giant fiber, intact giant nerve fiber, extruded axoplasm, and axoplasm-free giant nerve fiber sheaths. In 11 samples of stellar nerve devoid of giant fiber, weighing an average of 20.8 ± 2.3 mg ( s.e.m. ), 756 ± 91 pmol ACh and 8.65 ± 0.62 nmol of choline were found. The total ACh content of the largest fibre in this group (10 μ m in diameter), for a 5 cm length of nerve, is in the order of 0.16 pmol. The average wet weights of a single giant nerve fiber (270-420 μ m in diameter) and its separate components ( s.e.m .; in mg; number of fibers in parentheses) were: intact fiber, 4.58 ± 0.19 (25); extruded axoplasm, 3.38 ± 0.13 (20); sheaths, 1.21 ± 0.11 (16). The average ACh content per unit weight of sample was about 2-3 times higher in the sheaths (5-13 pmol-mg⁻¹) than in the axoplasm (2-4 pmol mg⁻¹), whereas the ACh concentrations estimated per unit volume of cellular water were about 40 times higher in the Schwann cell (107-222 μ m ) than in the axon (2-5 μ m ). These experimental findings establish the presence of ACh in the giant nerve fiber of S. sepioidea. They also indicate the Schwann cells themselves as the main source for the release of ACh, responsible for their long-lasting hyperpolarizations following the conduction of nerve impulse trains by the axon.     

FINE STRUCTURAL ALTERATIONS ASSOCIATED WITH VENOM ACTION ON SQUID GIANT NERVE FIBERS

(1) Block of conduction and marked increase in permeability of the squid giant axon, when surrounded by adhering small nerve fibers, is caused by the venoms of cottonmouth, ringhals, and cobra snakes and by phospholipase A (PhA). This phenomenon is associated with a marked breakdown of the substructure of the Schwann sheath into masses of cytoplasmic globules. Low concentrations of these agents which render the axons sensitive to curare cause less marked changes in the structure of the sheath. (2) Rattlesnake venom, the direct lytic factor obtained from ringhals venom, and hyaluronidase caused few observable changes in structure, correlating with the inability of these agents to increase permeability. (3) Cottonmouth venom did not alter the structure of giant axons freed of all adhering small nerve fibers. This is in agreement with previous evidence that the venom effects are due to an action of lysophosphatides liberated as a result of PhA action. Cetyltrimethylammonium chloride, a cationic detergent, produces effects that resemble those of venom and PhA. (4) The results provide evidence that PhA is the component of the venoms that is responsible for their effects. It also appears that the Schwann cell and possibly the axonal membrane are the major permeability barriers in the squid giant axon.     

PROTEIN COMPOSITION OF AXONS and MYELIN FROM RAT and HUMAN PERIPHERAL NERVES

Abstract— Proteins of rat and human peripheral nerves were studied in whole nerve homogenates and in purified myelin and axonal preparations of peripheral nerve. Both myelin and axonal fractions were obtained from desheathed and minced nerve segments by flotation and sedimentation, respectively, in 0.85 m -sucrose following hypotonic treatment. The purity of myelin and axonal preparations was confirmed by electron microscopic examination of pelleted material. Nerve proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at pH 8.3 and 7.4. Major protein bands of fresh whole nerve homogenates corresponded to polypeptide bands of either the purified myelin or axon preparations. The most prominent electrophoretic band in peripheral nerve was identified as a myelin glycoprotein with molecular weight of 27,000. The major polypeptides of axon preparations had molecular weights of 200,000, 150,000, 69,000, 55,000 and 27,000. The latter two proteins were believed to represent tubulin and residual major myelin protein, respectively. The three largest axonal polypeptides were believed to be derived from neurofilaments, which represented the predominant organelle of the purified axons. Collagen was also seen in whole nerve homogenates and in purified axons but could be distinguished by its metachromatic staining with Coomassie blue.     

热休克蛋白代谢过程中Hela细胞热耐受性的变化

HeLa细胞受热应激后,可产生一组热休克蛋白(HSP),其中HSP73/70产量最高,其合成呈现一定的规律性,受热后4h为其合成速率高峰,10h后明显减少,24h恢复正常。随着HSP合成的消失,正常蛋白质合成逐渐恢复。HSP73/70在细胞内分解遵循指数规律,其半衰期为49.9h。HSP合成及分解规律与细胞热耐受性的增加与消退基本吻合,提示二者之间存在着伴随关系,但是否存在量效关系乃至因果关系有待今后进一步探讨。