AXONAL TRANSPORT OF SELECTED PARTICLE-SPECIFIC ENZYMES IN RAT SCIATIC NERVE IN VIVO AND ITS RESPONSE TO INJURY

Abstract— Orthograde and retrograde axoplasmic transport of selected axonal organelles were examined by monitoring accumulation of enzyme activities residing in various types of particles proximal and distal to a ligature placed on rat sciatic nerve as a function of time after tying. Proximal to the tie, activity of acetylcholinesterase (AChE, EC 3.1.1.7; probably in small endoplasmic reticulum-like particles) accumulated for 2 days; then, during the next 5 days, the accumulation disappeared. Activities of glutamic dehydrogenase (GDH, EC 1.4.1.3) and monoamine oxidase (MAO, EC 1.4.3.4) (both located in mitochondria) accumulated steadily for 7 days. Accumulation of monoamine oxidase activity was more rapid than that of glutamic dehydrogenase during the first day or two. Acid phosphatase (acid P'tase, EC 3.1.3.2; in lysosomes) activity also accumulated throughout the week of observation. Accumulation of all four enzyme activities proximal to the ligature was blocked by nerve crush or subepineurial vinblastine injection 1 cm or more proximal to the site of the tie. Distal to the ligature, AChE activity accumulated early (14 h), and then gradually disappeared in the course of the week. MAO activity also accumulated, with a maximum at 2 days, and no further change thereafter. GDH activity, on the other hand, showed little accumulation during the first 2 days, but did appear in modest amounts at the end of the week. Distal accumulation of acid P'tase kept pace with proximal accumulation for the first day, and continued more slowly for another day, after which there was no further change. This system has been used to study the effects of axonal crush injury upon anterograde and retrograde axoplasmic transport. A tie applied at various times after injury, proximal to the site of injury, was used to show that orthograde transport of AChE was maintained for 1 day after tying, but at 2 days had fallen 50% or more, and within a week was down to 20–25% of control. At 3 days after injury retrograde transport of AChE activity was not different from the control. Orthograde transport of acid P'tase activity was depressed 35% by injury. Retrograde transport of acid P'tase was inhibited more than 50% both at 3 and at 7 days after injury. Transport of the mitochondrial enzymes was not measurably affected.     

Transported Enzymes in Sciatic Nerve and Sensory Ganglia of Rats Exposed to Maternal Antibodies Against Nerve Growth Factor

Abstract: The accumulations by axoplasmic transport of selected enzyme activities proximal and distal to a ligature placed on the sciatic nerve were monitored in rats exposed in utero to maternal antibodies to nerve growth factor (NGF) and in control rats. Littermates of the animals exposed to anti-NGF were shown elsewhere to have had a 70% reduction in the number of sensory neurons in dorsal root ganglia and a 90% reduction in number of neurons in superior cervical (sympathetic) ganglion. The accumulation of F^--sensitive acid phosphatase activity was depressed 75% both proximal and distal to the tie. Accumulation of F^--resistant acid phosphatase activity was depressed nearly 50% proximal to the tie. Distal accumulation of this activity did not occur in either group of rats. Accumulation of acetylcholinesterase activity was not affected. Proximal accumulation of glutamic dehydrogenase activity was depressed 30%. Distal accumulation of the activities of β-glucuronidase and hexokinase was depressed 50%. In the lumbar dorsal root ganglia, dry weight was reduced 40%, and the activities of peroxide-sensitive, F^--resistant acid phosphatase and of the mitochondrial enzymes hexokinase, glutamic dehydrogenase, glutamic-oxalacetic transaminase, and NAD-dependent isocitric dehydrogenase were all reduced a little more, 45–50% per ganglion. However, the activities of the lysosomal enzymes, F^--sensitive acid phosphatase and β-glucuronidase, of the peroxide-resistant, F^--resistant acid phosphatase, and of the mitochondrial enzyme glutaminase were all reduced about 60% per ganglion. The results of these measurements were interpreted to suggest that much, and perhaps all, of the F^--sensitive acid phosphatase activity in motion in peripheral nerve in rat is confined to sensory axons.     

Ganglioside Treatment of Streptozotocin-Diabetic Rats Prevents Defective Axonal Transport of 6-Phosphofructokinase Activity

This study measured axonal transport of 6-phosphofructokinase (PFK) and aldolase activities in the sciatic nerves of rats with short-term streptozotocin-induced diabetes. The diabetic rats showed deficits in anterograde (69% of controls; p less than 0.001) and retrograde (33% of controls; p less than 0.01) accumulations of PFK activity as well as its content per unit length of unconstricted sciatic nerve (86% of controls; p less than 0.05). There were no accumulation deficits in aldolase activity in the nerves of the diabetic rats, although the activity per unit length of unconstricted nerve was deficient (81% of controls; p less than 0.05). Treatment of diabetic rats with mixed bovine brain gangliosides (10 mg/kg of body weight/day, i.p.) did not affect the deficit in PFK activity in unconstricted nerve (84% of ganglioside-treated controls; p less than 0.01), but all the other defects in enzyme activities were prevented completely. The diabetic rats also showed a reduction of 7% (p less than 0.01) in sciatic nerve dry weight per unit length, which was prevented by ganglioside treatment. In contrast, the reduced motor nerve conduction velocity, accumulation of polyol pathway metabolites, and depletion of myo-inositol, characteristic of untreated short-term diabetes, were unaffected by ganglioside treatment.     

AXONAL TRANSPORT OF PHENYLETHANOLAMINE-N-METHYLTRANSFERASE IN TOAD SCIATIC NERVE

—The presence of phenylethanolamine-N-methyltransferase (EC 2.1.1.-) and dopamine-β-hydroxylase (EC 1.14.2.1) activities was demonstrated in the sciatic nerve of the toad, Bufo marinus. The rates of accumulation of phenylethanolamine-N-methyltransferase (PNMT) and dopamine-β-hydroxylase (DBH) proximal to a ligation of the sciatic nerve were studied. DBH accumulated proximal to the ligation at a more than 10-fold faster rate than PNMT. By measuring the rate of loss of enzyme activity distal to a ligation, an estimate of per cent clearance of each enzyme was made. Based on the per cent of enzyme activity free to move, the absolute transport rates for each enzyme were estimated to be: PNMT, 3.6 mm/24 h; DBH, 102 mm/24 h. PNMT activity (89 per cent) was recovered in the soluble fraction of sciatic nerve homogenates with no change occurring in the subcellular distribution of the enzyme proximal to ligations. In contrast, 43 per cent of DBH activity was found in the soluble fraction of sciatic nerve homogenates; but a disproportionate increase in paniculate DBH activity was found proximal to sciatic nerve ligations. Reduction of toad body temperature to 4°C resulted in a complete but totally reversible block of the axonal transport of both PNMT and DBH.     

AXONAL TRANSPORT OF CATECHOLAMINE SYNTHESIZING AND METABOLIZING ENZYMES

The rates of accumulation of the catecholamine synthesizing and metabolizing enzymes proximal to a ligation on the sciatic nerve of the rat were studied. Dopamine-β hydroxylase (EC 1.14.2.1) and tyrosine hydroxylase (EC 1.14.3a) accumulated at a similar rapid rate, and catechol-O-methyl-transferase (EC 2.1.1.6), choline acetyltransferase (EC 2.3.1.6) and monoamine oxidase (EC 1.4.3.4) accumulated at the same slow rate, whereas DOPA decarboxylase (EC 4.1.1.26) accumulated at an intermediate rate. Based on clearance of the rapidly accumulating enzymes, absolute flow rates were estimated to be: 106-167 mm/24 h for tyrosine hydroxylase; 138-185 mm/24 h for dopamine-β-hydroxylase; and 36-86 mm/24 h for DOPA decarboxylase. In contrast, the mean rate of transport of the slowly accumulating enzymes (monomine oxidase, catechol-O-methyltransferase and choline acetyltransferase) was approximately 3 mm/24 h. Colchicine and vinblastine completely blocked the axonal transport of both the rapidly and slowly transported enzymes. Studies of the subcellular distribution of each enzyme failed to confirm the suggestion that particulate enzymes are transported rapidly and soluble enzymes slowly. Our results suggest that the transport and inactivation of dopamine-β-hydroxylase, DOPA decarboxylase, and tyrosine hydroxylase are under different controls than monoamine oxidase and catechol-O-methyltransferase.     

Pathways of carbohydrate metabolism in peripheral nervous tissue. I. The contribution of alternative routes of glucose utilization in peripheral nerve and brain

The activities of enzymes of the glycolytic route, the pentose phosphate pathway, the tricarboxylic acid cycle and lipogenesis have been measured in rat sciatic nerve and brain. Parallel studies have been made of the utilization of 14 C-labelled glucose and pyruvate in these two tissues. Comparison of the enzyme profiles and flux through alternative routes was based on activity relative to the rate of glucose phosphorylation as measured by the rate of formation of 3H2O from [2-3H]glucose. The contributions of the pentose phosphate pathway and lipogenesis to glucose utilization were substantially higher in sciatic nerve than brain. The relatively high activity of transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2) suggested a special role for these enzymes in sciatic nerve.     

STUDIES ON AXOPLASMIC TRANSPORT OF INDIVIDUAL PROTEINS: 1–ACETYLCHOLINESTERASE (AChE) IN ACRYLAMIDE NEUROPATHY

Abstract— The axoplasmic transport rate and distribution of acetylcholinesterase (AChe, EC 3.1.1.7) was studied in the sciatic nerves of normal rats and those with a neuropathy due to acrylamide, by measuring the accumulation of the enzyme proximal to single and double ligatures. The single ligature experiments showed that the apparent transport rate of AChE was decreased in acrylamide neuropathy. The double ligature experiments indicated that only 8.1% of AChE was mobile in normal rat sciatic nerve. The mobility of the enzyme in acrylamide-treated rat sciatic nerves was altered to 11.8%. The absolute transport rate of AChE in normal rat sciatic nerve was 567 mm/24 h, and in acrylamide neuropathy it was decreased to 287 mm/24 h.
The amount of AChE activity transported in normal rat sciatic nerve was 2.64 μmol/24 h. The rats with acrylamide neuropathy showed a decrease in the amount of AChE activity moving in the orthograde direction (2.03 μmol/24 h).
The colchicine-binding properties of tubulin protein from sciatic nerves of normal and acrylamide-treated rats were studied. In rats with acrylamide neuropathy, a marked decrease of 75% in tubulin-colchicine binding was observed.     

Proteolysis of peripheral nerve myelin in acute experimental allergic neuritis

Proteolysis of peripheral nerve myelin was studied in rats with experimental allergic neuritis (EAN). In vitro measurements using rat sciatic nerve homogenate and denatured bovine myelin as a substrate showed two myelin specific enzyme activities at pH 3.8 (inhibited by pepstatin) and pH 5.8 (inhibited by PMSF) in the normal rat and newly appearing activities at pH 2.8 (inhibited by pepstatin) and pH 5.0 (not characterized) in the EAN rat. In EAN the proteolytic activity was not restricted to myelin substrate but degraded total sciatic nerve protein as well. Endogenous sciatic nerve protease at pH 5.8 did not significantly change in activity during the course of disease. On the contrary, activity of acid protease at pH 2.8 corresponded well to the disease. Myelin degradation in EAN, therefore, appears to be mainly due to exogenous non-tissue protease.Abbreviations EAN experimental allergic neuritis - EDTA ethylenediaminetetraacetic acid - HBM hydroxymercuro benzoate - PLP proteolipid protein - PMSF phenylmethylsulfonyl fluoride - PNS peripheral nervous system - SDS sodium dodecylsulfate - TCA trichloroacetic acid This work is part of the M.D. thesis of R. B.     

RELATIONSHIP BETWEEN THE RATE OF AXOPLASMIC TRANSPORT AND SUBCELLULAR DISTRIBUTION OF ENZYMES INVOLVED IN THE SYNTHESIS OF NOREPINEPHRINE

The net rate of proximo-distal transport of tyrosine hydroxylase, dopamine β-hydroxylase, DOPA decarboxylase and choline acetyltransferase was determined by measuring the accumulation of these enzymes proximal to a ligature of the rat sciatic nerve. The rate of accumulation was constant for at least 12 h. For the enzymes involved in the biosynthesis of norepinephrine the rate of transport was correlated to their subcellular distribution and a close correlation between these two parameters was found. Dopamine β-hydroxylase, an enzyme mainly localized in the particulate fraction of the sciatic nerve, showed the fastest rate of transport (1·94 mm/h) whereas DOPA decarboxylase, exclusively located in the high-speed supernatant fluid, gave the slowest (0·63 mm/h) rate of transport. Tyrosine hydroxylase, predominantly located in the non-particulate fraction of the sciatic nerve was transported much slower (0·75 mm/h) than dopamine β-hydroxylase but still significantly (P < 0.005) faster than DOPA decarboxylase. The subcellular distribution of dopamine β-hydroxylase in ganglia did not differ significantly (0·45 > P > 0·40) from that in the sciatic nerve, but in nerve endings a greater proportion of dopamine β-hydroxylase was localized in particulate fractions. Tyrosine hydroxylase and DOPA decarboxylase were found exclusively in the non-particulate fractions of ganglia. In the nerve endings of the effector organs a small but consistent portion of tyrosine hydroxylase was found in particulate fractions, whereas DOPA decarboxylase was exclusively localized in the high-speed supernatant fluid.     

Multiple forms of monoamine oxidase in rabbit platelets.

Rabbit platelets were found to contain both types A and B MAO activities. The specific enzymatic activity of rabbit platelet MAO was higher for the substrate serotonin than for phenylethylamine. The K_m's for rabbit platelet MAO indicated that the MAO-B enzyme was similar to human platelet MAO and that both MAO-A and MAO-B enzymes in the rabbit platelet are similar to the corresponding forms in the rabbit brain. The drugs clorgyline and deprenyl confirmed the existence of types A and B MAO in the platelet and furthermore indicated that the type A form accounted for approximately 90% of the total enzymatic activity. Amitriptyline at low (micromolar) concentrations selectively inhibited MAO-B activity in both rabbit platelets and brain.     

Transport of axonal enzymes in surviving segments of frog sciatic nerve

Redistribution of axonal enzymes as a function of time in vitro was studied in an unbranched segment of frog sciatic nerve. Cholinesterase activity moved peripherally at a rate of 99 mm/day and centrally at 19 mm/day. One-quarter of the total nerve content of the enzyme was estimated to be in motion, one-eighth in each direction. Mitochondrial enzymes (hexokinase and glutamic dehydrogenase) moved peripherally at 20–31 mm/day, centrally at 11–20 mm/day. Only 10% of the total content of these mitochondrial enzymes was in motion. No movement of choline acetylase or 6-phosphogluconic dehydrogenase activity was seen even after 4 days in vitro. However, in a 12 day in vivo experiment choline acetylase moved toward the periphery at a rate of 0.34 mm/day. After a day or so in vitro the distal accumulations of cholinesterase and glutamic dehydrogenase decreased, with a concomitant and quantitatively equivalent increase in enzyme activities at the proximal end of the nerve. It is postulated that during incubation a mechanism for reversing the direction of flow develops in the peripheral stump of the nerve. Vinblastine inhibited central and peripheral flow of both cholinesterase and glutamic dehydrogenase. Movement of cholinesterase was not affected by ouabain, thalidomide, or phenobarbital, nor by K⁺ excess (110 mM) or absence.     

CHOLESTEROL, CHOLESTEROL ESTERS AND CHOLESTEROL ESTERASE IN THE SCIATIC NERVE DURING DEVELOPMENT OF THE CHICK

Abstract— Changes in the contents of cholesterol and cholesterol ester and in the activity of cholesterol esterase (EC 3.1.1.13) in the sciatic nerve of the chick during development were measured. The ratio of cholesterol ester/cholesterol was an inverse function of the activity of the esterase (i.e. when the ratio was high, the activity of the enzyme was low, and vice versa). The synthesis of cholesterol esterase was inhibited after addition of actinomycin D both in ovo and in vitro.     

SEPARATION OF APPARENT MULTIPLE FORMS OF HUMAN BRAIN CHOLINE ACETYLTRANSFERASE BY ISOELECTRIC FOCUSING

Abstract— Choline acetyltransferase (acetyl-CoA: choline O -acetyl transferase; EC 2.3.1.6; ChAc) purified from human brain (basal ganglia) and sciatic nerve were separated into apparent multiple enzyme forms by the method of isoelectric focusing (pH gradient 3-10) on acrylamide gel. A preparative separation of enzyme forms of human brain was accomplished by the column method, by using a sucrose gradient. When each separated form was re-electrofocused, only a portion of the ChAc activity was observed in its original pH region while more than one-half of the recovered activity for each fraction appeared at pH 7.8-8. Gel filtration and kinetic studies of separated forms indicated that the more acidic forms might be aggregates, while more basic forms might be configurational isomers. Human ChAc of sciatic nerve did not exhibit acidic forms on electrofocusing, but otherwise yielded an electrofocusing profile similar to that of human brain. ChAc of rabbit brain and sciatic nerve each exhibited only a single form at pH 7.1 ± 0.2. Although ChAc differs among species, the enzyme of brain and sciatic nerve of the same species cannot be clearly distinguished by electrofocusing.     

Essential sulfhydryl groups of rat liver monoamine oxidase.

The inhibition by some thiol reagents of partly purified mitochondrial monoamine oxidase (MAO) (EC 1.4.3.4) from rat liver was studied, and the molar content of sulfhydryl groups in the enzyme determined. Sodium nitroprusside and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) inhibited the enzyme, apparently reversibly, while sodium arsenite was not inhibitory. Concentrations of the respective inhibitors causing 50% inhibition after 15 min of preincubation with the enzyme at pH 7.0 and 37 degrees C are 5.80 times 10(-4) M and 4.35 times 10(-5) M. The thiol compounds cysteine, dithiothreitol, and 2-mercaptoethanol did not inhibit MAO. The average number of sulfhydryl groups per mole of enzyme, determined by reaction with DTNB, increased from 3.6 +/- 0.2 freely reacting sulfhydryl groups (n = 4) to 18.4 to total sulfhydryl groups (n = 2) on denaturation with 8 M urea.     

Modification of substrate-inhibitor affinities of human platelet monoamine oxidase B in vitro

The rate of benzylamine utilization by monoamine oxidase (MAO)-B from human blood platelets was 2-4 times higher than that for octopamine. Both activities were inhibited 100% by 10(-7) M deprenyl (a specific MAO-B inhibitor) and were not affected by clorgyline (a specific MAO-A inhibitor) or by polyclonal antibodies to MAO-A. The preincubation of platelet MAO-B with purified MAO-A from mitochondrial membranes of human placenta resulted in appearance of excess octopamine activity. This additional activity was not precipitated by antibodies to MAO-A or inhibited by deprenyl but was inhibited by clorgyline. Incubation of the MAO-A preparation from placenta at 45 degrees C for 15 min before its preincubation with MAO-B caused 50% loss of both activities. Protease inhibitors had no effect on the modification of MAO. These data indicate that MAO-A or a factor tightly bound to it can modify MAO-B yielding a form of the enzyme with both MAO-A and MAO-B substrate and inhibitor affinities and MAO-B immunospecificity.     

猕猴神经系统器官蛋白水解酶种类和性质研究

实验以太行山猕猴为材料,用活性电脉（Ｇ－ＰＡＧＥ）方法分析研究了神经系统中蛋白水解酶的种类、活性及ｐＨ依赖性,结果表明：（１）神经系统各部分均具有３１、３０、２９ｋｕ三种酸性蛋白水解酶;（２）９４ｋｕ的中性蛋不解酶普遍存在于神经系统各器官中;（３）在中性和碱性条件下,坐骨神经中蛋白水解酶活性较强,其余部分生微弱。     

Transport of Muscarinic Cholinergic Marker Protein Activities in Regenerating Sciatic Nerve of Rat

The transport characteristics of choline acetyltransferase (ChAT; EC 2.3.1.6), acetylcholinesterase (AChE; EC 3.1.1.7), and the muscarinic acetylcholine receptors (mAChR) were studied in perineurally sutured, regenerating rat sciatic nerve. At different times after repair, the sciatic nerve was ligated for 24 h, and the activities of the cholinergic marker proteins, as well as the binding capacity, were measured proximally and distally from the ligature. The number of bidirectionally transported receptors increased linearly up to 5 months postoperatively (6.1-33.6% and 5.6-25.6% of the control level proximal and distal to the ligature, respectively). The quantity of anterogradely transported ChAT reached a plateau 3 months postoperatively (74.9% of the control level), whereas the retrogradely transported enzyme was then only 34.7% of the control value. The activity of AChE increased linearly during nerve regeneration, and exceeded the control level after 4 months (121.0% and 63.7% proximally and distally, respectively). The data indicate that the altered bidirectional transport of cholinergic marker proteins may be monitored quantitatively during nerve regeneration. This method might be suitable for studies of the nerve regeneration process.     

FAST AXOPLASMIC TRANSPORT OF ACETYLCHOLINESTERASE IN MAMMALIAN NERVE FIBRES

Abstract— Acetylcholinesterase (acetylcholine acetyl-hydrolase, EC 3.1.1.7) is carried down mammalian nerve fibres by the fast axoplasmic transport system. This conclusion was derived from experiments involving the ligation of cat sciatic nerves at two sites placed 83.5 mm apart. The enzyme accumulated in segments of nerve proximal to the upper ligation in a linear fashion over a period of at least 20 h. At approximately 5 h the accumulation of enzyme ceased in the nerve segment proximal to the distal ligation within the isolated length of nerve, an observation indicating that the portion of AChE free to move within the isolated nerve had been depleted during this period of time. The freely moving fraction of AChE was estimated to be 15% of the total enzyme activity present in the nerve (10% in the proximo-distal direction and 5% in the retrograde direction). The rate of AChE downflow (as estimated from the intercept of the curve plotting accumulation with the line denoting when depletion started) was 431 mm/day within a 95% confidence interval of 357–543 mm/day. In view of the variability, our results demonstrated that AChE was being carried by the fast axoplasmic transport system, which in earlier studies was estimated to have a characteristic rate close to 410 mm/day.
An accumulation of AChE was also found on the distal side of the ligations that represented a movement of AChE in the distal-proximal direction in the fibres. This retrograde transport was smaller in amount (about one-half) than the proximo-distal rate of transport, or close to 220 mm/day. The rate of AChE transport was discussed in relation to the 'transport filament' hypothesis of fast axoplasmic transport.     

ENZYMES CATALYSING ETHANOL METABOLISM IN NEURAL AND SOMATIC TISSUES OF THE RAT

Abstract— The enzymes catalysing ethanol metabolism, alcohol dehydrogenase (EC 1.l.1.1) and aldehyde dehydrogenase (EC 1.2.1.3), were assayed in a variety of neural and somatic tissues of the rat, the human counterparts of which are known to be vulnerable to excessive ethanol. The activity of alcohol dehydrogenase was assayed by the coupled oxidation of ethanol and reduction of lactaldehyde, a method which we have recently found to be sufficiently sensitive and specific to measure the relatively low levels of activity in whole brain. Detectable activities of these enzymes were found in peripheral nerve, skeletal muscle, retina, optic nerve and various regions of brain, as well as in a variety of non-neural tissues. The levels of the enzymic activities in all tissues were markedly lower than those of liver, but probably sufficient to perform a local function in the metabolism of ethanol or other endogenous substrates. The activity of alcohol dehydrogenase in the various tissues, like that of liver, was confined to the cytosol and exhibited kinetic properties and responses to inhibitors almost identical to those of the liver enzyme. We consider the results to be consistent with the hypothesis that the pathological effects of alcohol may be related, at least in part, to local mechanisms for the metabolism of alcohol.