Acetylcholine Receptors : Distribution and extrajunctional density in rat diaphragm after denervation correlated with acetylcholine sensitivity

Using ¹²⁵iodine-labeled α-bungarotoxin (α-BGT-¹²⁵I) and quantitative radioautography, we have studied the time-course of the change in acetylcholine (ACh) receptor distribution and density occurring in rat diaphragm after denervation. In innervated fibers, ACh receptors are localized at the neuromuscular junction and the extrajunctional receptor density is less than five receptors per square micrometer. The extrajunctional receptor density begins to increase between 2 and 3 days after denervation and increases approximately linearly to 1695 receptors/µm² at 14 days, subsequently decreasing to 529 receptors/µm² at 45 days. We have isolated plasma membranes from rat leg muscles at various times after denervation and find that the change in concentration of ACh receptors in the membranes measured by α-BGT-¹²⁵I binding and scintillation counting follows a time-course similar to the change in ACh receptor density measured radioautographically. Furthermore, we have correlated extrajunctional ACh receptor density measured by radioautography with extrajunctional ACh sensitivity measured by iontophoretic application of ACh and intracellular recording and find that the log of ACh receptor density is related to 0.53 times the log of ACh sensitivity. These results are discussed in terms of the electrophysiological experiments on the ACh receptor and the recent, more biochemical approaches to the study of ACh receptor control and function.     

Sequence of Age-Associated Changes to the Mouse Neuromuscular Junction and the Protective Effects of Voluntary Exercise

Loss of connections between motor neurons and skeletal muscle fibers contribute to motor impairment in old age, but the sequence of age-associated changes that precede loss of the neuromuscular synapse remains uncertain. Here we determine changes in the size of neuromuscular synapses within the tibialis anterior muscle across the life span of C57BL/6J mice. Immunofluorescence, confocal microscopy and morphometry were used to measure the area occupied by nerve terminal synaptophysin staining and postsynaptic acetylcholine receptors at motor endplates of 2, 14, 19, 22, 25 and 28month old mice. The key findings were: 1) At middle age (14-months) endplate acetylcholine receptors occupied 238±11 µm² and nerve terminal synaptophysin 168±14 µm² (mean ± SEM). 2) Between 14-months and 19-months (onset of old age) the area occupied by postsynaptic acetylcholine receptors declined 30%. At many endplates the large acetylcholine receptor plaque became fragmented into multiple smaller acetylcholine receptor clusters. 3) Between 19- and 25-months, the fraction of endplate acetylcholine receptors covered by synaptophysin fell 21%. By 28-months, half of the endplates imaged retained ≤50 µm² area of synaptophysin staining. 4) Within aged muscles, the degree to which an endplate remained covered by synaptophysin did not depend upon the total area of acetylcholine receptors, nor upon the number of discrete receptor clusters. 5) Voluntary wheel-running exercise, beginning late in middle-age, prevented much of the age-associated loss of nerve terminal synaptophysin. In summary, a decline in the area of endplate acetylcholine receptor clusters at the onset of old age was followed by loss of nerve terminal synaptophysin from the endplate. Voluntary running exercise, begun late in middle age, substantially inhibited the loss of nerve terminal from aging motor endplates.     

STEREOLOGICAL ANALYSIS OF MAMMALIAN SKELETAL MUSCLE : I. Soleus Muscle of the Adult Guinea Pig

A quantitative analysis of the volumes, surface areas, and dimensions of the ultrastructural components in the soleus muscle fibers of the guinea pig was made by using point counting methods of stereology. Muscle fibers have structural orientation (anisotropy) and have spatial gradients of the structures within the fiber; therefore the standard stereological methods were modified where necessary. The entire analysis was repeated at two section orientations to test the modifications and identical results obtained from both. The volume of lipid droplets was 0.20 ± 0.06% (mean ± standard error, n = 5 animals) and the nuclei volume was 0.86 ± 0.20% of the fiber volume. The total mitochondrial volume was 4.85 ± 0.66% of the fiber volume with about one-third being found in an annulus within 1 µm of the sarcolemma. The mitochondrial volume in the remaining core of the fiber was 3.6 ± 0.4%. The T system has a volume of 0.14 ± 0.01% and a surface area of 0.064 ± 0.005 µm²/µm³ of the fiber volume. The surface area of the sarcolemma is 0.116 ± 0.013 µm²/µm³ which is twice the T system surface area. The volume of the entire sarcoplasmic reticulum is 3.52 ± 0.33% and the surface area is 0.97 ± 0.09 µm²/µm³. The sarcoplasmic reticulum is composed of the terminal cisternae whose volume is 1.04 ± 0.19% and surface area is 0.24 ± 0.05 µm²/µm³. The tubules of the sarcoplasmic reticulum in the I band and A band have volumes of 1.97 ± 0.24% and 0.51 ± 0.08%, and the surface areas of the I and A band reticulum are 0.56 ± 0.07 µm²/µm³ and 0.16 ± 0.04 µm²/µm³, respectively. The Z line width, myofibril and fiber diameters were measured.     

Laminin-induced Clustering of Dystroglycan on Embryonic Muscle Cells: Comparison with Agrin-induced Clustering

The effect of laminin on the distribution of dystroglycan (DG) and other surface proteins was examined by fluorescent staining in cultures of muscle cells derived from Xenopus embryos. Western blotting confirmed that previously characterized antibodies are reactive in Xenopus. In control cultures, αDG, βDG, and laminin binding sites were distributed as microclusters (<1 μm² in area) over the entire dorsal surface of the muscle cells. Treatment with laminin induced the formation of macroclusters (1–20 μm²), accompanied by a corresponding decline in the density of the microclusters. With 6 nM laminin, clustering was apparent within 150 min and near maximal within 1 d. Laminin was effective at 30 pM, the lowest concentration tested. The laminin fragment E3, which competes with laminin for binding to αDG, inhibited laminin-induced clustering but did not itself cluster DG, thereby indicating that other portions of the laminin molecule in addition to its αDG binding domain are required for its clustering activity. Laminin-induced clusters also contained dystrophin, but unlike agrin-induced clusters, they did not contain acetylcholine receptors, utrophin, or phosphotyrosine, and their formation was not inhibited by a tyrosine kinase inhibitor. The results reinforce the notion that unclustered DG is mobile on the surface of embryonic muscle cells and suggest that this mobile DG can be trapped by at least two different sets of molecular interactions. Laminin self binding may be the basis for the laminin-induced clustering.     

Penetration of Rhizopus oligosporus into Soybeans in Tempeh

Histological observations were made on the penetration of hyphae of Rhizopus oligosporus into soybean cotyledons in tempeh, an Indonesian soybean food. Hyphal penetrations averaged one per 1,400 μm² (±390 μm²) on the curved (outer) cotyledon surface and one per 1,010 μm² (±340 μm²) on the flat (inner) one. Hyphae infiltrated to a depth of 742 μm, or about 25% of the average width of a soybean cotyledon. This previously unreported degree of penetration offers partial explanation for the rapid physical and chemical changes in soybeans during tempeh fermentation.     

Electrical Properties and Acetylcholine Sensitivity of Singly and Multiply Innervated Avian Muscle Fibers

Membrane constants and distribution of acetylcholine (ACh) receptors were determined for multiply innervated fibers of the anterior latissimus dorsi (ALD) and singly innervated fibers of the posterior latissimus dorsi (PLD) muscles of 3–6 month old chickens. The values of the various membrane constants were: length constant, 1.78 mm (mean) in ALD, 0.68 mm in PLD; time constant, 35 msec in ALD, 3.7 msec in PLD; transverse membrane resistance, 4388 Ω cm² in ALD, 561 Ω cm² in PLD; and membrane capacitance, 8.2 µF/cm² in ALD, 7.0 µF/cm² in PLD. Peaks of ACh sensitivity occurred at intervals of ca. 740 µ on ALD fibers with a low sensitivity remaining between peaks. Only one peak of ACh sensitivity was detected on PLD fibers. The maximum ACh sensitivity found was 5 ± 4 mv/ncoul for fibers of the ALD and 77 ± 60 mv/ncoul for fibers of the PLD. The distance over which this sensitivity fell to 0.1 was ca. 225 µ in the ALD and 140 µ in the PLD. The membranes of these two muscle fiber types differ widely regarding some electrical properties and the disposition of ACh-sensitive receptor sites.     

Denervation causes fiber atrophy and myosin heavy chain co-expression in senescent skeletal muscle

Although denervation has long been implicated in aging muscle, the degree to which it is causes the fiber atrophy seen in aging muscle is unknown. To address this question, we quantified motoneuron soma counts in the lumbar spinal cord using choline acetyl transferase immunhistochemistry and quantified the size of denervated versus innervated muscle fibers in the gastrocnemius muscle using the in situ expression of the denervation-specific sodium channel, Nav_1.5, in young adult (YA) and senescent (SEN) rats. To gain insights into the mechanisms driving myofiber atrophy, we also examined the myofiber expression of the two primary ubiquitin ligases necessary for muscle atrophy (MAFbx, MuRF1). MN soma number in lumbar spinal cord declined 27% between YA (638±34 MNs×mm⁻¹) and SEN (469±13 MNs×mm⁻¹). Nav_1.5 positive fibers (1548±70 μm²) were 35% smaller than Nav_1.5 negative fibers (2367±78 μm²; P<0.05) in SEN muscle, whereas Nav_1.5 negative fibers in SEN were only 7% smaller than fibers in YA (2553±33 μm²; P<0.05) where no Nav_1.5 labeling was seen, suggesting denervation is the primary cause of aging myofiber atrophy. Nav_1.5 positive fibers had higher levels of MAFbx and MuRF1 (P<0.05), consistent with involvement of the proteasome proteolytic pathway in the atrophy of denervated muscle fibers in aging muscle. In summary, our study provides the first quantitative assessment of the contribution of denervation to myofiber atrophy in aging muscle, suggesting it explains the majority of the atrophy we observed. This striking result suggests a renewed focus should be placed on denervation in seeking understanding of the causes of and treatments for aging muscle atrophy.     

Cyclic variations in nitrogen uptake rate in soybean plants

Uptake of NO₃⁻ by nonnodulated soybean plants (Glycine max L. Merr. cv Ransom) growing in flowing hydroponic culture at 22 and 14°C root temperatures was measured daily during a 31-day growth period. Ion chromatography was used to determine removal of NO₃⁻ from solution during each 24-hour period. At both root-zone temperatures, rate of NO₃⁻ uptake per plant oscillated with a periodicity of 3 to 5 days. The rate of NO₃⁻ uptake per plant was consistently lower at 14°C than 22°C. The lower rate of NO₃⁻ uptake at 14°C during the initial 5 to 10 days was caused by reduced uptake rates per gram root dry weight, but with time uptake rates per gram root became equal at 14 and 22°C. Thereafter, the continued reduction in rate of NO₃⁻ uptake per plant at 14°C was attributable to slower root growth.     

Temperature and Photoperiod Influence Trichome Density and Sesquiterpene Content of Lycopersicon hirsutum f. hirsutum

Resistance to Colorado potato beetle in a clone of Lycopersicon hirsutum f. hirsutum L. is attributed to the presence of the sesquiterpene zingiberene in the type VI leaf trichomes; however, both day/night temperature regimen and photoperiod affect zingiberene content and trichome density. In short days (SD), zingiberene content per trichome is more than 3-fold greater than in long days. In SD, trichome density per unit leaf surface is 2-fold greater at 25/20°C (day/night) than at either 30/25°C or 20/15°C, thus indirectly influencing zingiberene content per cm². In long days, temperature regimen had little effect on either trichome density or zingiberene content, although trichome density was greater than or equal to that in SD.     

Estimation of the fructose diphosphatase-phosphofructokinase substrate cycle in the flight muscle of Bombus affinis

1. Substrate cycling of fructose 6-phosphate through reactions catalysed by phosphofructokinase and fructose diphosphatase was estimated in bumble-bee (Bombus affinis) flight muscle in vivo. 2. Estimations of substrate cycling of fructose 6-phosphate and of glycolysis were made from the equilibrium value of the ³H/¹⁴C ratio in glucose 6-phosphate as well as the rate of ³H release to water after the metabolism of [5-³H,U-¹⁴C]glucose. 3. In flight, the metabolism of glucose proceeded exclusively through glycolysis (20.4μmol/min per g fresh wt.) and there was no evidence for substrate cycling. 4. In the resting bumble-bee exposed to low temperatures (5°C), the pattern of glucose metabolism in the flight muscle was altered so that substrate cycling was high (10.4μmol/min per g fresh wt.) and glycolysis was decreased (5.8μmol/min per g fresh wt.). 5. The rate of substrate cycling in the resting bumble-bee flight muscle was inversely related to the ambient temperature, since at 27°, 21° and 5°C the rates of substrate cycling were 0, 0.48 and 10.4μmol/min per g fresh wt. respectively. 6. Calcium ions inhibited fructose diphosphatase of the bumble-bee flight muscle at concentrations that were without effect on phosphofructokinase. The inhibition was reversed by the presence of a Ca²⁺-chelating compound. It is proposed that the rate of fructose 6-phosphate substrate cycling could be regulated by changes in the sarcoplasmic Ca²⁺ concentration associated with the contractile process.     

Bacterial Biomass, Metabolic State, and Activity in Stream Sediments: Relation to Environmental Variables and Multiple Assay Comparisons

Bacterial biomass, metabolic condition, and activity were measured over a 16-month period in the surface sediments of the following four field sites with differing dissolved organic matter regimes: a woodlot spring seep, a meadow spring seep, a second-order stream, and a third-order stream. Total bacterial biomass was measured by lipid phosphate and epifluorescence microscopic counts (EMC), and viable biomass was measured by ¹⁴C most probable number, EMC with 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride reduction, and ATP. Bacterial metabolic condition was determined from the percentage of respiring cells, poly-β-hydroxybutyrate concentrations, and adenylate energy charge. Activity measures included ¹⁴C-lipid synthesis, ³²P-phospholipid synthesis, the rate of uptake of algal lysate dissolved organic carbon, and respiration, from which biosynthesis was calculated (dissolved organic carbon uptake corrected for respiration). Total bacterial biomass (from EMC) ranged from 0.012 to 0.354 μg of C/mg of dry sediment and was usually lowest in the third-order stream. The percentage of cells respiring was less than 25% at all sites, indicating that most bacteria were dormant or dead. Adenylate energy charge was measured only in the third-order stream and was uniformly low. Poly-β-hydroxybutyrate concentrations were greater in the woodlot spring seep than in the second- and third-order streams. Uptake of algal lysate dissolved organic carbon ranged from undetectable levels to 166 mg of C · m⁻² · h⁻¹. Little community respiration could be attributed to algal lysate metabolism. Phospholipid synthesis ranged from 0.006 to 0.354 pmol · mg of dry sediment⁻¹ · h⁻¹. Phospholipid synthesis rates were used to estimate bacterial turnover at the study sites. An estimated 375 bacterial generations per year were produced in the woodlot spring seep, and 67 per year were produced in the third-order stream.     

Enzymes in cancer: Asparaginase from chicken liver

1. A procedure for partial purification of asparaginase from chicken liver is presented. 2. The bulk of the enzyme is located in the soluble fraction of chicken liver. 3. Molecular weights of chicken-liver asparaginase and of the guinea-pig serum enzyme, estimated by gel filtration, were 306000 and 210000 respectively. The Michaelis constants (K_m) at 37° and pH8·5 were 6·0×10⁻⁵m and 7·2×10⁻⁵m respectively. 4. At 50° the chicken-liver enzyme was moderately stable, some activity being lost by aggregation; in dilute electrolyte solutions the activity rapidly diminished. 5. The anti-lymphoma effect of guinea-pig serum in mice carrying the 6C3HED tumour was confirmed. Chicken-liver asparaginase also showed an effect but in this case the enzyme preparation had to be administered repeatedly. 6. Guinea-pig serum asparaginase was stable for several days in mouse blood, after intraperitoneal injection, whereas chicken-liver asparaginase rapidly disappeared. 7. Aspartic acid β-hydrazide was shown to be a competitive inhibitor of chicken-liver asparaginase with K_i approx. 5·6×10⁻⁴m. In mice it produced an anti-lymphoma effect, as reported previously.     

Effects of Heavy Strength Training on Running Performance and Determinants of Running Performance in Female Endurance Athletes

Purpose

The purpose of the current study was to investigate the effects of adding strength training to normal endurance training on running performance and running economy in well-trained female athletes. We hypothesized that the added strength training would improve performance and running economy through altered stiffness of the muscle-tendon complex of leg extensors.

Methods

Nineteen female endurance athletes [maximal oxygen consumption (VO_2max): 53±3 ml∙kg^-1∙min^-1, 5.8 h weekly endurance training] were randomly assigned to either normal endurance training (E, n = 8) or normal endurance training combined with strength training (E+S, n = 11). The strength training consisted of four leg exercises [3 x 4–10 repetition maximum (RM)], twice a week for 11 weeks. Muscle strength, 40 min all-out running distance, running performance determinants and patellar tendon stiffness were measured before and after the intervention.

Results

E+S increased 1RM in leg exercises (40 ± 15%) and maximal jumping height in counter movement jump (6 ± 6%) and squat jump (9 ± 7%, p < 0.05). This was accompanied by increased muscle fiber cross sectional area of both fiber type I (13 ± 7%) and fiber type II (31 ± 20%) in m. vastus lateralis (p < 0.05), with no change in capillary density in m. vastus lateralis or the stiffness of the patellar tendon. Neither E+S nor E changed running economy, fractional utilization of VO_2max or VO_2max. There were also no change in running distance during a 40 min all-out running test in neither of the groups.

Conclusion

Adding heavy strength training to endurance training did not affect 40 min all-out running performance or running economy compared to endurance training only.     

Inhibition of glucose phosphate isomerase by metabolic intermediates of fructose

1. Purified rabbit-muscle and -liver glucose phosphate isomerase, free of contaminating enzyme activities that could interfere with the assay procedures, were tested for inhibition by fructose, fructose 1-phosphate and fructose 1,6-diphosphate. 2. Fructose 1-phosphate and fructose 1,6-diphosphate are both competitive with fructose 6-phosphate in the enzymic reaction, the apparent K_i values being 1·37×10⁻³−1·67×10⁻³m for fructose 1-phosphate and 7·2×10⁻³−7·9×10⁻³m for fructose 1,6-diphosphate; fructose and inorganic phosphate were without effect. 3. The apparent K_m values for both liver and muscle enzymes at pH7·4 and 30° were 1·11×10⁻⁴−1·29×10⁻⁴m for fructose 6-phosphate, determined under the conditions in this paper. 4. In the reverse reaction, fructose, fructose 1-phosphate and fructose 1,6-diphosphate did not significantly inhibit the conversion of glucose 6-phosphate into fructose 6-phosphate. 5. The apparent K_m values for glucose 6-phosphate were in the range 5·6×10⁻⁴−8·5×10⁻⁴m. 6. The competitive inhibition of hepatic glucose phosphate isomerase by fructose 1-phosphate is discussed in relation to the mechanism of fructose-induced hypoglycaemia in hereditary fructose intolerance.     

A MORPHOMETRIC STUDY ON THE NEXUS OF RAT CARDIAC MUSCLE

A morphometric study of the nexus of rat cardiac muscle was carried out. The nexus surface of one intercalated disk of one 15 µm thick fiber is found to range between 47 µm² and 94 µm², the latter value taking into account the maximal underestimation caused by tangential sectioning. Dividing the lower, minimal value by the surface of the observed subunits (90 Å periodicity), one obtains for one intercalated disk 6.7 x 10⁵ subunits, each of them assumed to be permeated by a central pore. These pores are thought to be equivalent to intercellular channels in a recently proposed model. Taking our morphometric and recently reported physiological values, this model is examined for its consistency with a low resistance pathway between cardiac muscle cells.     

Effects of Abiotic Factors on Acetylene Reduction by Cyanobacteria Epiphytic on Moss at a Subantarctic Island

Acetylene reduction (AR) rates by cyanobacteria epiphytic on a moss at Marion Island (46°54′ S, 37°45′ E) increased from −5°C to a maximum at 25 to 27°C. Q₁₀ values between 0 and 25°C were between 2.3 and 2.9, depending on photosynthetic photon flux density. AR rates declined sharply at temperatures above the optimum and were lower at 35°C than at 0°C. Photosynthetic photon flux density at low levels markedly influenced AR, and half of the maximum rate occurred at 84 μmol m⁻² s⁻¹, saturation occurring at ca. 1,000 μmol m⁻² s⁻¹. Higher photosynthetic photon flux density levels decreased AR rates. AR increased up to the highest sample moisture content investigated (3,405%), and the pH optimum was between 5.9 and 6.2. The addition of P, Co, and Mo, individually or together, depressed AR.     

ELECTRON MICROSCOPE RADIOAUTOGRAPHY AS A QUANTITATIVE TOOL IN ENZYME CYTOCHEMISTRY : II. The Distribution of DFP-Reactive Sites at Motor Endplates of A Vertebrate Twitch Muscle

The distribution of diisopropylfluorophosphate (DFP)-sensitive enzyme sites at the neuromuscular junction was determined quantitatively by electron microscope radioautography after incubation of muscle fragments in DFP-³H. Most of the sensitive sites were located in the subneural apparatus at a concentration of 90,000 sites per µ³ of cleft tissue or 12,000 sites per µ² of postjunctional membrane surface area. A considerable concentration is also present in the teloglial cap. It has previously been demonstrated (Rogers et al., 1966) that one-third of the DFP-sensitive sites at the endplate can be reactivated by pyridine-2-aldoxime methiodide (2-PAM)—a compound which selectively reactivates phosphorylated acetylcholinesterase. In the present study, it was found that this ratio of 1:2 holds also on a fine-structural level. Muscle mast cells were found to have a heavy concentration of bound DFP.     

Unilateral Muscle Overuse Causes Bilateral Changes in Muscle Fiber Composition and Vascular Supply

Unilateral strength training can cause cross-transfer strength effects to the homologous contralateral muscles. However, the impact of the cross-over effects on the muscle tissue is unclear. To test the hypothesis that unilateral muscle overuse causes bilateral alterations in muscle fiber composition and vascular supply, we have used an experimental rabbit model with unilateral unloaded overstrain exercise via electrical muscle stimulation (E/EMS). The soleus (SOL) and gastrocnemius (GA) muscles of both exercised (E) and contralateral non-exercised (NE) legs (n = 24) were morphologically analyzed after 1w, 3w and 6w of EMS. Non-exercised rabbits served as controls (n = 6). After unilateral intervention the muscles of both E and NE legs showed myositis and structural and molecular tissue changes that to various degrees mirrored each other. The fiber area was bilaterally smaller than in controls after 3w of E/EMS in both SOL (E 4420 and NE 4333 µm² vs. 5183 µm², p<0.05) and GA (E 3572 and NE 2983 µm² vs. 4697 µm², p<0.02) muscles. After 6w of E/EMS, the percentage of slow MyHCI fibers was lower than in controls in the NE legs of SOL (88.1% vs. 98.1%, p<0.009), while the percentage of fast MyHCIIa fibers was higher in the NE legs of GA (25.7% vs. 15.8%, p = 0.02). The number of capillaries around fibers in the E and NE legs was lower (SOL 13% and 15%, respectively, GA 25% and 23%, respectively, p<0.05) than in controls. The overall alterations were more marked in the fast GA muscle than in the slow SOL muscle, which on the other hand showed more histopathological muscle changes. We conclude that unilateral repetitive unloaded overuse exercise via EMS causes myositis and muscle changes in fiber type proportions, fiber area and fiber capillarization not only in the exercised leg, but also in the homologous muscles in the non-exercised leg.     

Inhibition of purine phosphoribosyltransferases of Ehrlich ascites-tumour cells by 6-mercaptopurine

1. The formation of adenosine 5′-phosphate, guanosine 5′-phosphate and inosine 5′-phosphate from [8-¹⁴C]adenine, [8-¹⁴C]guanine and [8-¹⁴C]hypoxanthine respectively in the presence of 5-phosphoribosyl pyrophosphate and an extract from Ehrlich ascites-tumour cells was assayed by a method involving liquid-scintillation counting of the radioactive nucleotides on diethylaminoethylcellulose paper. The results obtained with guanine were confirmed by a spectrophotometric assay which was also used to assay the conversion of 6-mercaptopurine and 5-phosphoribosyl pyrophosphate into 6-thioinosine 5′-phosphate in the presence of 6-mercaptopurine phosphoribosyltransferase from these cells. 2. At pH 7·8 and 25° the Michaelis constants for adenine, guanine and hypoxanthine were 0·9 μm, 2·9 μm and 11·0 μm in the assay with radioactive purines; the Michaelis constant for guanine in the spectrophotometric assay was 2·6 μm. At pH 7·9 the Michaelis constant for 6-mercaptopurine was 10·9 μm. 3. 25 μm-6-Mercaptopurine did not inhibit adenine phosphoribosyltransferase. 6-Mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 4·7 μm) and hypoxanthine phosphoribosyltransferase (K_i 8·3 μm). Hypoxanthine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 3·4 μm). 4. Differences in kinetic parameters and in the distribution of phosphoribosyltransferase activities after electrophoresis in starch gel indicate that different enzymes are involved in the conversion of adenine, guanine and hypoxanthine into their nucleotides. 5. From the low values of K_i for 6-mercaptopurine, and from published evidence that ascites-tumour cells require supplies of purines from the host tissues, it is likely that inhibition of hypoxanthine and guanine phosphoribosyltransferases by free 6-mercaptopurine is involved in the biological activity of this drug.