兔肌3-磷酸甘油脱氢酶的提纯及性质研究

目的:研究兔肌3-磷酸甘油脱氢酶的分离纯化方法及其酶学性质,为测定血清甘油三酯所用酶联试剂的开发提供试验基础和理论依据。方法:通过硫酸铵分级沉淀、DEAE-Sepharose、Blue-Sepharose和羟磷灰石纯化兔肌3-磷酸甘油脱氢酶,利用凝胶过滤和梯度PAGE(5%～15%)法测定酶分子量,采用常规酶学动力学分析方法,考察pH、温度、底物浓度以及部分金属离子与有机化合物对酶促反应的影响。结果 纯化后的兔肌3-磷酸甘油脱氢酶经PAGE(12%)分析为单一条带;酶分子量为115～122 kDa;酶最适温度45℃,最适pH 9;酸碱稳定范围pH6～9,低于45℃时热稳定性好;最适条件下,以3-磷酸甘油和NAD⁺为底物,测得酶的K_m分别为7.4×10^-3mol/L和1.47×10^-4mol/L;Ba²⁺、Mn²⁺、Fe²⁺、Al³⁺、Cu²⁺、Ni²⁺、Ag⁺、Hg²⁺、NaN₃、EDTA对酶有不同程度的抑制作用,Mg²⁺、Ca²⁺、Co²⁺、Zn²⁺有一定程度的激活作用,其中Co²⁺和Zn²⁺对酶的激活作用能达到200%以上,有机化合物NaF对酶的活性没有影响。     

Glycerol Phosphate Dehydrogenase, Glucose-6-Phosphate Dehydrogenase, and Lactate Dehydrogenase: Activities in Oligodendrocytes, Neurons, Astrocytes, and Myelin Isolated from Developing Rat Brains

Abstract: Glycerol phosphate dehydrogenase (GPDH), glucose-6-phosphate dehydrogenase (G6PDH), and lactate dehydrogenase (LDH) activities were determined in Oligodendrocytes, neurons, and astrocytes isolated from the brains of developing rats. The activity of each enzyme was significantly lower in both neurons and astrocytes than in Oligodendrocytes. The GPDH activity in Oligodendrocytes increased more than 4-fold during development, and at 120 days cells of this type had 1.4-fold the specific activity of forebrain homogenates. The G6PDH activities in Oligodendrocytes from 10-day-old rats were 1.4-fold the activities in the forebrain homogenates. The activities of this enzyme in Oligodendrocytes were progressively lower at later ages, such that at 120 days the cells had 0.8 times the specific activities of homogenates. The Oligodendrocytes had 0.6 times the homogenate activities of LDH at 10 days, and this ratio had decreased to 0.2 by 120 days. These enzymes were also measured in myelin isolated from 20-, 60-, and 120-day-old rats. By 120 days the specific activities of G6PDH and LDH in myelin were <8% of the respective activities in homogenates. The GPDH activity in myelin was, however, at least 20% the specific activity in the homogenates, even in the oldest animals. It is proposed that LDH could be used as a marker for oligodendroglial cytoplasm in subfractions of myelin and in myelin-related membrane vesicles.     

Decrease of Brain Phospholipid Synthesis in Free-Moving n-3 Fatty Acid Deficient Rats

Abstract: The autoradiographic method with [¹⁴C]-docosahexaenoic acid ([¹⁴C]22:6 n-3) was used to determine whether a diet deficient in n-3 fatty acids, inducing a decrease in 22:6 n-3 circulating level, was associated with changes in local rates of phospholipid synthesis in the rat brain. As compared with rats fed a normal diet (peanut plus rapeseed oil), a n-3 fatty acid deficiency [peanut oil group (P group)] induced a generalized decrease (?35 to ?76%) of 22:6 n-3 incorporation rates into phospholipids in all the regions examined. This effect was confirmed by using [³H]22:6 n-3 infusion by biochemical analysis and quantifications corrected for the contribution of docosahexaenoate derived from lipid store recycling to the unesterified pool, taken as the precursor pool for phospholipid synthesis in the whole brain. In normal or n-3 fatty acid-deficient rats, the values of the brain-to-plasma 22:6 n-3 specific activity ratio (Ψ) were similar (0.03), indicating that a considerable endogenous source of 22:6 n-3 (97%), likely derived from phospholipid degradation, dilutes the specific activity of the tracer coming from plasma. Using the specific activity of 22:6 n-3 in plasma instead of brain would thus lead to a gross underestimation of the rate of phospholipid synthesis. The results also demonstrate that the pattern of ¹⁴C or ³H distribution in brain lipids was not modified by the n-3 fatty acid-deficient diet. The major lipids labeled were phospholipids, particularly phosphatidylethanolamine. Nevertheless, the unesterified 22:6 n-3 concentrations in plasma and brain were significantly reduced (eight- and threefold, respectively) in the P group. In addition, the proportion of 22:6 n-3 in the brain total lipid fraction, total phospholipids, and phosphatidylcholine, -ethanolamine, and -serine was significantly decreased in n-3 fatty acid-deficient rats. This was partially compensated for by an increase in the 22:5 n-6 level. These results are discussed in relation to the limitation of 22:6 n-3 use to quantify, by the quantitative autoradiographic method, changes in local rates of phospholipid synthesis in rat brain.     

Glycerol Phosphate Dehydrogenase in Developing Chick Retina and Brain

Abstract: The development of cytoplasmic glycerol phosphate dehydrogenase (GPDH) activity in chick neural retina is compared with that in brain. GPDH converts dihydroxyacetone phosphate to glycerol 3-phosphate, an intermediate in phospholipid synthesis. The enzyme is known to be under corticosteroid control in rat brain and spinal cord (but not muscle or liver) and in primary oligodendrocyte cultures. It has not been previously studied in the eye. In chick brain the GDPH specific activity rises fivefold from the early embryo to the adult, with nearly all the increase occurring between embryonic day 14 and hatching. This time course correlates well with the known maturation of chick adrenal cortex (which produces corticosteroids). On the other hand, in chick retina the GPDH specific activity remains at a low basal level throughout development. Furthermore, adult rat and beef retinas show much lower enzyme activity than do the corresponding brain tissues. GPDH can be induced precociously by hydrocortisone in embryonic chick brain from days 12 through 16, both in the intact embryo and in tissue culture; however, GPDH is not at all inducible in chick retina. The developmental increase in chick brain GPDH can be correlated qualitatively with myelin formation, as shown by luxol fast blue staining, whereas no myelin is seen in retina at any age. Our results are consistent with recent immunocytochemical studies demonstrating that GPDH in rat brain is associated with myelin-producing oligodendroglial cells, absent in retina. In comparison, another glial enzyme, glutamine synthetase (GS), known to be inducible in both chick brain and retina, is localized in brain astrocytes and retinal Müller cells.     

Regulation of NADP-Dependent Glyceraldehyde 3-Phosphate Dehydrogenase Activity in Spinach Chloroplasts*

Activation of NAD(P)-glyceraldehyde 3-phosphate dehydrogenase (NADP-GAPDH, EC 1.2.1.13) can be achieved in isolated chloroplasts in the light, or in the dark upon addition of dithiothreitol (DTT) and/or 3-phosphoglycerate plus ATP. Activation in darkened chloroplasts is only partial with DTT or 3-phosphoglycerate plus ATP alone, but complete when both effectors are added. In the light, full activation is only achieved upon addition of ATP. The time-course of activation appears to depend upon the actual concentration of 1,3-bisphosphoglycerate (1,3bisPGA) inside the chloroplasts. The K_a values for 1,3bisPGA are in the same range as has been determined for the purified enzyme, namely around 20 μM for the dark form (in the absence of DTT) and around 1 μM for the light form or in the presence of DTT. In contrast, the K_a value for ATP is 1 to 2 mM for both the oxidized and the reduced enzyme forms. The observed activation of NADP-GAPDH is strongly paralleled by an increase of 3PGA, and consequently of 1,3bisPGA in the illuminated chloroplast, while the ATP level remains constant or declines. Activation by 1,3bisPGA is accompanied by dissociation of the 600 kDa form to the 150 kDa form, while reduction alone does not induce a shift in molecular mass as documented by fast gel filtration on Superdex 200. Thus partial activation by DTT in the dark is due to an increased activity of the 600 kDa form, while the activation state in the light is the result of a partial conversion of the 600 kDa form into the more active 150 kDa form. The principle of this activation is a fast reduction of the enzyme by the ferredoxin/thioredoxin system, resulting in a lowered K_avalue for 1,3bisPGA, and thus adjusting the properties of the enzyme to the stromal 1,3bisPGA level. The occurrence of a 300 kDa oligomer mainly during inactivation has also been observed. From these results a model is constructed that describes the reversible interconversion of various activation and aggregation states of NADP-GAPDH as observed upon light/dark transitions in isolated spinach chloroplasts.     

Cotransport of Glyceraldehyde-3-Phosphate Dehydrogenase and Actin in Axons of Chicken Motoneurons

1.To study proteins transported with actin in axons, we pulse-labeled motoneurons in the chicken sciatic nerve with [³⁵S]methionine and, 1–20 days later, isolated actin and its binding proteins by affinity chromatography of Triton soluble nerve extracts on DNase I–Sepharose. The DNase I-purified proteins were electrophoresed on two-dimensional gels and the specific activity of the radioactively labeled protein spots was estimated by fluorography.2.In addition to actin, which binds specifically to DNase I, a small number of other proteins were labeled, including established actin monomer binding proteins and a protein of 36 kDa and pI 8.5. On the basis of its molecular mass, pI, amino acid composition, and immunostaining, the unrecognized protein was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH).3.The high-affinity binding of GAPDH to actin was confirmed by incubation of Triton-soluble nerve extracts with either mouse anti-GAPDH (or antiactin) and indirect immunomagnetic separation with Dynabeads covalently linked to sheep anti-mouse antibody. Analysis by one-dimensional gel electrophoresis and immunoblotting showed that actin and GAPDH were the main proteins isolated by these methods.4.Analysis of labeled nerves at 12 and 20 days after pulse labeling showed that GAPDH and actin were transported at the same rate, i.e., 3–5 mm/day, which corresponds to slow component b of axonal transport. These proteins were not associated with rapidly transported proteins that accumulated proximal to a ligation 7 cm from the spinal cord 9 hr after injection of radioactivity.5.Our results indicate that GAPDH and actin are transported as a complex in axons and raise the possibility that GAPDH could act as a chaperone for monomeric actin, translocating it to intraaxonal sites for exchange with or assembly into actin filaments. Alternatively, actin could be involved in translocating and anchoring GAPDH to specialized sites in axons and nerve terminals that require a source of ATP by glycolysis.     

Adenosine Receptors in Myelin Fractions and Subtractions: The Effect of the Agonist (R)-Phenylisopropyladenosine on Myelin Membrane Microviscosity

In this article the existence of A1 adenosine receptors and the absence of A2 adenosine receptors in myelin membranes purified from pig brain white matter are demonstrated. The characterization of (R)-[3H]phenylisopropyladenosine ([3H]R-PIA) binding to purified myelin fractions was performed. The distribution of high- and low-affinity species of the A1 adenosine receptor was different in heavy, medium, and light myelin. The fluidity of myelin subfractions and of pig brain cortical membranes was estimated; the microviscosity of heavy myelin (5.4 poises) and of cortical membranes (5.1 poises) was similar and less than that of medium (7.8 poises) and light (8.2 poises) myelin. It was also demonstrated that the agonist R-PIA modifies the microviscosity of myelin membranes and that the degree of modification depends on the fluidity of the membrane assayed. These results suggest that adenosine receptors may have an important role in the functionality of myelin membranes.     

Ornithine Decarboxylase Induction by Glucocorticoids in Brain and Liver of Adrenalectomized Rats

Abstract: Ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, was measured in the brain and the liver of adrenalectomized rats after an acute S.C. treatment with glucocorticoids. The effects of corticosterone and dexamethasone were compared in three brain areas, the cerebral cortex, hippocampus, and cerebellum. These structures have similar concentrations of cytosolic glucocorticoid receptor, as measured by an in vitro exchange assay using a specific glucocorticoid ligand, [³H]RU 26988, but contain different amounts of mineralocorticoid receptor. Corticosterone and dexamethasone increased ODC activity in the liver and brain areas in a dose dependent manner, dexamethasone being more active than corticosterone in all tissues. Moreover, estradiol, progesterone, and testosterone were inactive. Aldosterone, at high doses, increased brain ODC activity. Glucocorticoids, selected for their weak binding, or lack of binding to the mineralocorticoid receptor, were tested and found to be highly active in inducing brain and liver ODC, thus showing that ODC induction by steroids is specific for glucocorticoids. These results are among the first to suggest biochemically a central action of glucocorticoids following an acute treatment and confirm that the brain is a glucocorticoid target organ.     

Angiotensin-Converting Enzyme Activity Is Reduced in Brain Microvessels of Spontaneously Hypertensive Rats

Abstract: Angiotensin-converting enzyme (ACE) activity in brain microvessels of spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) controls was measured. Cerebral microvessels, prepared from the cerebral cortices by the albumin flotation and glass bead filtration technique, were free of neuronal and glial elements. ACE activity in brain microvessels of SHR was lower than that of WKY. A Woolf-Augustinsson-Hofstee plot showed that the reduction of the enzyme activity in SHR was due to a 30% decrease in V_max without any change in K_m for substrate. The decrease of ACE activity in brain micro-vessels of SHR may indicate an impairment of the central renin-angiotensin system and may be related to cerebral microvascular dysfunctions occurring in hypertension.     

Regulation of Brain and Cerebrospinal Fluid Calcium by Brain Barrier Membranes Following Vitamin D-Related Chronic Hypo- and Hypercalcemia in Rats

Male Fischer-344 rats, 21 days old, were fed diets containing 0 (LOD), 2,200 (CONT), or 440,000 (HID) international units of vitamin D3 per kilogram for 12 weeks. [Ca] was measured in plasma, CSF, brain, and choroid plexus. In addition, 45Ca and 36Cl transfer coefficients (KCa and KCl) for uptake from blood into CSF and brain were determined. Although plasma ionized [Ca]s in LOD and HID rats were 50% and 136%, respectively, of values in CONT animals, CSF and brain [Ca]s ranged from only 85% to 110% of respective CONT values. Choroid plexus [Ca] was increased by 37% after HID diet, but was decreased only 10% after LOD. KCa values at CSF, parietal cortex, and pons-medulla were negatively correlated with plasma ionized [Ca], whereas KCl values at CSF and brain were not different between the diet groups. The findings demonstrate that central nervous system [Ca] is maintained during chronic hypo- or hypercalcemia by saturable transport of Ca at brain barrier membranes. This transport does not seem to involve modulation by 1,25-dihydroxyvitamin D3.     

Glyceraldehyde-3-Phosphate Dehydrogenase Activity and F-Actin Associations in Synaptosomes and Postsynaptic Densities of Porcine Cerebral Cortex

1. Glyceraldehyde-3-phosphate dehydrogenase (G3PD) is a glycolytic enzyme that has also been implicated in a wide variety of functions within neurons. Because of the well-documented role of G3PD as an actin-binding protein, we sought evidence for a G3PD–actin complex in synaptosomes and postsynaptic densities (PSDs).2. We have shown G3PD association with 0.5-m synaptosomal particles by immunofluorescence as similarly demonstrated for actin (Toh et al., Nature 264:648–650, 1976). An immunoblot analysis also showed G3PD and actin to be enriched in synaptosomes. Further analysis of subcellular fractions from synaptosomes showed the PSD but not the synaptosomal plasma membranes to be enriched in G3PD and actin.3. Highest levels of G3PD catalytic activity were found in synaptosomes and PSDs. Although synaptosomes showed significant activity for phosphoglyceratekinase (PGK), an enzyme in sequence with G3PD for ATP production in the glycolytic pathway, no such activity was detected in the PSD fraction.4. Our studies indicate that a G3PD–actin complex may exist at the synapse. A physical association of G3PD with endogenous F-actin in synaptosomes and PSDs was demonstrated by combined phalloidin shift velocity sedimentation/immunoblot studies. By this approach, synaptosomal G3PD–actin complexes were also found to be significantly less dense than the PSD G3PD–actin complexes.5. G3PD and PGK catalytic activity in synaptosomes suggests a role in glycolysis, as well as actin binding, in the presynaptic terminals. On the other hand, the high levels of G3PD activity in PSDs but lack of PGK activity suggests that G3PD is involved in nonglycolytic functions, such as actin binding and actin filament network organization.     

Monitoring the Effect of a Tryptophan Load on Brain Indole Metabolism in Freely Moving Rats by Simultaneous Cerebrospinal Fluid Sampling and Brain Dialysis

Rats were given L-tryptophan, 50 mg/kg i.p., and its concentration in the CNS was monitored in individual freely moving animals using repeated sampling of cisternal CSF and concurrent striatal dialysis. The 5-hydroxytryptamine metabolite 5-hydroxyindoleacetic acid (5-HIAA) was also measured. Results were compared with changes of central tryptophan and 5-HIAA concentrations in brains of rats killed at various times after administration of L-tryptophan, 50 mg/kg i.p. Tryptophan changes in CSF were proportionate to those in whole brain and followed essentially identical time courses. Results for the striatal dialysate and whole striatum also paralleled each other. Similarly, results for 5-HIAA showed proportionality between CSF and brain and between dialysate and striatum. The data obtained were used to determine pharmacokinetic data for individual rats, i.e., areas under curves for both tryptophan and 5-HIAA and half-lives for the decline of tryptophan. Kinetic parameters varied considerably from rat to rat. However, mean half-lives for tryptophan in CSF, brain, dialysate, and striatum were all comparable. Results in general show the value of repeated CSF sampling and intracerebral dialysis for concurrent monitoring of changes of indole metabolism in the whole brain and a specific brain region, respectively. The methods should be suitable for the continuous monitoring of changes of central transmitter metabolism in parallel with observation of behavior following environmental or dietary changes or drug administration. They also should be of use in the investigation of drug kinetics in the CNS.     

Determination of Brain 5-Hydroxytryptamine Turnover in Freely Moving Rats Using Repeated Sampling of Cerebrospinal Fluid

A simple technique is described for repeated sampling of cerebrospinal fluid (CSF) from the freely moving rat and its use in the determinations of 5-hydroxytryptamine (5-HT) turnover validated. A catheter, constructed from polyethylene tubing (PP10) was implanted via a cranial approach into the cisterna magna and x-ray studies confirmed that the catheter avoided the cerebellum. 5-HT turnover was determined from the rate of rise of 5-hydroxyindoleacetic acid (5-HIAA) in both CSF and brain following an injection of probenecid (200 mg/kg i.p.). Concentrations of 5-HIAA, 5-HT and tryptophan were determined by high pressure liquid chromatography. Turnover values for individual rats were obtained using CSF samples. After p-chlorophenylalanine treatment (when brain 5-HT was depleted by 43%) 5-HT turnover values obtained were comparably reduced whether determined from CSF (-67%) or brain (-74%). Thus differences of rat brain 5-HT turnover are proportionately reflected by CSF measurements. The method for sampling of CSF should be applicable in a wide range of pharmacological and physiological situations.     

[3H] Diazepam Displacing Activity in Human Cerebrospinal Fluid

Pooled human cerebrospinal fluid was separated by Sephadex G-50 chromatography. The presence of three peaks, A, B and C, was demonstrated by monitoring absorbance at 254 and 280 nm. All peaks showed [3H]diazepam displacing activity in the membrane receptor test. Peak B was further separated on Bio-Gel P-4. At least two major fractions free of salt and GABA in the molecular weight range of approximately 700--3600 were shown to displace [3H]diazepam in the receptor test. This activity was enhanced by a factor of 3 in the presence of 10 microM-GABA.     

Rat Brain Glyceraldehyde-3-Phosphate Dehydrogenase Interacts with the Recombinant Cytoplasmic Domain of Alzheimer's β-Amyloid Precursor Protein

Abstract: Abundant senile plaques are a histological hallmark in the brain of Alzheimer's disease patients. Such plaques consist of, among many other constituents, aggregated βA4 amyloid peptide. This peptide is derived from an amyloid precursor protein (APP) by irregular proteolytic processing and is considered to be involved in the development of Alzheimer's disease. To study possible interactions of brain proteins with 0A4 amyloid or other fragments of APP, βA4 amyloid and βA4 amyloid extended to the C-terminus of APP were recombinantly produced as fusion proteins termed "Amy" and "AmyC," respectively. Using Amy and AmyC affinity chromatography, a 35-kDa protein from rat brain was isolated that bound tightly to AmyC but not to Amy, thus indicating an interaction of the protein with the C-terminus of APP. This 35-kDa protein was identified as the glycolytic enzyme gIyceraldehyde-3-phosphate dehydrogenase (GAPDH). Binding of GAPDH to AmyC but not to Amy was confirmed by gel filtration. Although AmyC slightly reduced the V_max of GAPDH, the same reduction was observed in the presence of Amy. These findings suggest that the interaction of the cytoplasmic domain of APP with GAPDH is unlikely to influence directly the rate of glycolysis but may serve another function.     

盐生杜氏藻甘油-3-磷酸脱氢酶的分离纯化及其特性的研究

利用ＰＥＧ分级,ＤＥＡＥ离子交换层析,ＢｌｕｅＳｅｐｈａｒｏｓｅ拟亲和层析,ＭｏｎｏＱ离子交换层析等手段,分离纯化盐生杜氏藻（Ｄｕｎａｌｉｅｌａｓａｌｉｎａ（Ｄｕｎａｌ）Ｔｅｏｄ．）甘油三磷酸（Ｇ３Ｐ）脱氢酶（ＥＣ１．１．１．８）,得到比活为１２．６Ｕ／ｍｇ的电泳纯的酶,并对此酶的生化特性进行了研究。４％～２０％非变性聚丙烯酰胺梯度凝胶电泳测得全酶分子量约为２７０ｋＤ,ＳＤＳＰＡＧＥ表明该酶只有一种分子量约为６５ｋＤ的亚基,据此推测该酶应为同四聚体。酶催化磷酸二羟丙酮（ＤＨＡＰ）还原的最适ｐＨ值为７．５,催化Ｇ３Ｐ脱氢的最适ｐＨ值为１０。该酶对４个底物还原型辅酶Ⅰ（ＮＡＤＨ）,二磷酸吡啶核苷酸（ＤＨＡＰ）,辅酶Ⅰ（ＮＡＤ）,Ｇ３Ｐ的表观Ｋｍ值分别为６３μｍｏｌ／Ｌ,２７２μｍｏｌ／Ｌ,１．５３ｍｍｏｌ／Ｌ,６．５２ｍｍｏｌ／Ｌ。该酶在保存过程中易失活。ＮＡＤＨ能降低酶失活的速度,而ＮＡＤ则不然。低浓度ＮａＣｌ对酶略有保护作用,但高浓度ＮａＣｌ加快酶的失活,且浓度越高效应越明显。     

Phospholipid and Ganglioside Composition in Rat Brain After Chronic Intake of Ethanol

Abstract: A total of 18 60-day-old male Wistar rats were divided into three groups of six animals each. One group was fed a basal diet containing high levels of protein, fat, carbohydrate, vitamins, and minerals and separately a solution of 25% sucrose-32% ethyl alcohol (wt/vol). A second group was offered water as the only drinking fluid and a similar solid diet, except that carbohydrate replaced ethanol isocalorically. A third group was maintained on the basal diet ad libitum . All groups of animals were killed in a sober state after 6 months of chronic ethanol treatment and lipid analyses were performed on brain homog-enates. Chronic treatment of the animals with ethanol produces statistically significant modification of the phospholipid and ganglioside patterns in rat brain. A statistically significant decrease of the total phospholipid content and of some of the investigated fractions, i.e., phos-phatidylcholine and phosphatidylserine, as well as an increase of phosphatidylinositol were observed. Chronic alcohol consumption was associated with a statistically significant increase in the total amount of ganglioside in rat brain. An increase in most of the investigated ganglioside fractions was indicated but the difference was statistically significant only for trisialoganglioside GT_1b. The amount of disialoganglioside GD_1a in these brains was decreased after chronic intake of ethanol.     

Rapid Communication Guanylyl Cyclase Activity in Rat Brain Myelin and White Matter

Abstract: Purified myelin from rat brainstem was found to have an appreciable level of guanylyl cyclase activity, as seen in the formation of 3',5'-cyclic GMP from [³H]GTP at a rate ∼45% that of whole brainstem. Freshly isolated myelin from pooled rat brain-stems was incubated with GTP in an appropriate mixture. This gave rise to 29.9 ± 3.6 pmol of 3',5'-cyclic GMP/mg of protein/min measured by HPLC and a similar result (26.7 ± 2.6 pmol/mg/min) with ¹²⁵l-3',5'-cyclic GMP radioimmunoassay. The latter method applied to the reaction product from whole brainstem gave a value of 56.6 ± 3.4 pmol/mg/min. In analyzing brainstem products by HPLC we observed in most trials concurrent formation of a second radiolabeled product that comigrated with 2',3'-cyclic GMP but that, on further examination, proved not to be that product. Its identity remains unknown.