HIGH-RESOLUTION AUTORADIOGAPHY : II. The Problem of Resolution

The resolution obtainable in electron microscopic autoradiographs, using a photographic emulsion consisting of a monolayer of silver bromide crystals, was investigated theoretically and experimentally. The expected distribution of exposed crystals around a point source was calculated from the geometry of the preparation and from the range distribution of the beta particles emitted by tritium. From such a distribution an autoradiographic resolution of the order of 1000 A can be predicted. From the point source distribution, the expected distribution of grains around bacteriophages labeled with tritium was calculated. This distribution was also measured experimentally in electron microscopic autoradiographs of bacteriophages T-2 labeled with thymidine-H³. The two distributions agreed closely. It was also verified, using the nuclear region in thin cross-sections of Bacillus subtilis labeled with thymidine-H³, that resolutions of the same order were obtained for extended sources. It was concluded that an autoradiographic resolution of 1000 A could be achieved with a presently available commercial emulsion, although emulsions with finer grains might be desirable in some circumstances.     

PROTEIN SYNTHESIS,STORAGE, AND DISCHARGE IN THE PANCREATIC EXOCRINE CELL : An Autoradiographic Study

The synthesis, intracellular transport, storage, and discharge of secretory proteins in and from the pancreatic exocrine cell of the guinea pig were studied by light- and electron microscopical autoradiography using DL-leucine-4,5-H³ as label. Control experiments were carried out to determine: (a) the length of the label pulse in the blood and tissue after intravenous injections of leucine-H³; (b) the amount and nature of label lost during tissue fixation, dehydration, and embedding. The results indicate that leucine-H³ can be used as a label for newly synthesized secretory proteins and as a tracer for their intracellular movements. The autoradiographic observations show that, at ∼5 minutes after injection, the label is localized mostly in cell regions occupied by rough surfaced elements of the endoplasmic reticulum; at ∼20 minutes, it appears in elements of the Golgi complex; and after 1 hour, in zymogen granules. The evidence conclusively shows that the zymogen granules are formed in the Golgi region by a progressive concentration of secretory products within large condensing vacuoles. The findings are compatible with an early transfer of label from the rough surfaced endoplasmic reticulum to the Golgi complex, and suggest the existence of two distinct steps in the transit of secretory proteins through the latter. The first is connected with small, smooth surfaced vesicles situated at the periphery of the complex, and the second with centrally located condensing vacuoles.     

Arginine utilization by Chlorella autotrophica and Chlorella saccharophila

Chlorella saccharophila can utilize the amino acids arginine, glutamate. ornithine and proline as sole sources of nitrogen for growth. By comparison C. autotrophica utilized only arginine and ornithine. Following osmotic shock of Chlorella autotrophica from 50 to 150% artificial seawater rapid synthesis of proline (the main osmoregulatory solute in this alga) occurred in cells grown on arginine or citrulline. However, little proline synthesis occurred in ornithine-grown cells. Distribution of radiolabelled carbon from [¹⁴C]-arginine assimilation following osmotic shock of C. autotrophica agrees with the following pathway of arginine utilization: arginine→citrulline→ornithine→glutamate semialdehyde→pyrroline-5-carboxylate→proline. These 4 steps are catalysed by arginine deiminase (EC 3.5.3.6), citrullinase (EC 3.5.1.20), ornithine transaminase (EC 2.6.1.13) and pyrroline-5-carboxylate reductase (EC 1.5.1.2), respectively. Of these 4 enzymes, only arginine deiminase and pyrroline-5-carboxylate reductase were detected in the crude extract of the 2 Chlorella species. Arginine deiminase did not require specific cations for optimal activity. The deimi-nase showed maximal activity at pH 8.0 and followed Michaelis-Menten kinetics with an apparent K_m for L-arginine of 0.085 m M for the C. autotrophica enzyme and 0.097 m M for that of C. saccharophila. The activity of arginine deiminase was not influen-ced by growing C. saccharophila on arginine. Ornithine competitively inhibited arginine deiminase with an apparent K, of 2.4 m M for the C. autotrophica enzyme, and 3.8 m M for that of C. saccharophila . Arginine utilization by Chlorella is discussed in relation to that of other organisms.     

Solubilization of Sodium Channel from Human Brain

[3H]Tetrodotoxin binds to a single class of receptor sites in homogenates of human brain with a KD of 9.1 nM at 0 degree C and a maximal binding capacity of 5.9 pmol/mg of protein. This tetrodotoxin receptor has been solubilized, and several parameters influencing the efficiency of this critical step have been studied. Treatment of brain membranes with 2% (wt/vol) Nonidet P-40 solubilizes up to 38% of the tetrodotoxin receptor sites. The duration of this solubilization step must not exceed 15 min at an optimal pH of 6.8. The binding activity is most stable when exogenous phosphatidylcholine is added to the soluble receptor with a phosphatidylcholine/detergent ratio of 1:5.     

Zinc, a novel structural element found in the family of bacterial adenylate kinases.

The adk gene from Bacillus stearothermophilus was cloned and overexpressed in Escherichia coli under the control of the lac promoter. The primary structure of B. stearothermophilus adenylate kinase exhibited 76% identity with the enzyme from Bacillus subtilis, 60% identity with the enzyme from Lactococcus lactis, and 42% identity with the enzyme from E. coli. The most striking property of the adenylate kinase from B. stearothermophilus is the presence of a structural zinc atom bound to four cysteines in a zinc finger-like fashion. The ability to coordinate zinc is predicted also for a number of other isoforms of bacterial adenylate kinases. Furthermore, the tightly bound metal ion contributes to the high thermodynamic stability of adenylate kinase from B. stearothermophilus.     

Neuromuscular development following tetrodotoxin-induced inactivity in mouse embryos

Developmental aspects of the neuromuscular system in mouse embryos chronically paralyzed in utero with tetrodotoxin (TTX) between embryonic days 14 and 18 were studied using biochemical and histological methods. The number of lumbar spinal motoneurons (MNs) was higher in inactive embryos than in controls suggesting a decreased motoneuron cell death. In association with the increase in MN number, choline acetyltransferase activity was significantly increased in both spinal cord and peripheral synaptic sites. Paralyzed muscles exhibited a decreased number of mature myofibers and the nuclei were centrally located. Creatine kinase activity was greatly decreased and total acetylcholine receptor and receptor cluster numbers per myofiber were significantly increased in paralyzed muscles. A similar pattern of changes occurs in the neuromuscular system of the mutant mouse muscular dysgenesis (mdg). However, in contrast to the mdg mutant, tetrodotoxin-treated muscles were similar to controls in their innervation pattern, in the ultrastructural aspects of the excitation–contraction coupling system (i.e., dyads and triads) and in the extent of dihydropyridine binding. Thus, neuromuscular inactivity is not sufficient to impair the pattern of muscle innervation or the appearance of either the triadic junctions or dihydropyridine receptors. These results indicate that alterations of dihydropyridine binding sites and triads in muscular dysgenesis cannot be accounted for by inactivity but rather must reflect a more primary defect involving the structural gene(s) regulating the development of one or more aspects of muscle differentiation.     

Subcellular distribution of carbonic anhydrase in Solanum tuberosum L. leaves

The intracellular compartmentation of carbonic anhydrase (CA; EC 4.2.1.1), an enzyme that catalyses the reversible hydration of CO₂ to bicarbonate, has been investigated in potato (Solanum tuberosum L.) leaves. Although enzyme activity was mainly located in chloroplasts (87% of total cellular activity), significant activity (13%) was also found in the cytosol. The corresponding CA isoforms were purified either from chloroplasts or crude leaf extracts, respectively. The cytosolic isoenzyme has a molecular mass of 255 000 and is composed of eight identical subunits with an estimated M _r of 30000. The chloroplastic isoenzyme (M _r 220000) is also an octamer composed of two different subunits with M _r estimated at 27 000 and 27 500, respectively. The N-terminal amino acid sequences of both chloroplastic CA subunits demonstrated that they were identical except that the M _r-27 000 subunit was three amino acids shorter than that of the M _r-27 500 subunit. Cytosolic and chloroplastic CA isoenzymes were found to be similarly inhibited by monovalent anions (Cl^–, I^–, N ₃ ^- and NO ₃ ^- ) and by sulfonamides (ethoxyzolamide and acetozolamide). Both CA isoforms were found to be dependent on a reducing agent such as cysteine or dithiothreitol in order to retain the catalytic activity, but 2-mercaptoethanol was found to be a potent inhibitor. A polyclonal antibody directed against a synthetic peptide corresponding to the N-terminal amino acid sequence of the chloroplastic CA monomers also recognized the cytosolic CA isoform. This antibody was used for immunocytolocalization experiments which confirmed the intracellular compartmentation of CA: within chloroplasts, CA is restricted to the stroma and appears randomly distributed in the cytosol.Abbreviations BSA bovine serum albumin - CA carbonic anhydrase - PMSF phenylmethylsulphonyl fluoride - BAM benzamidine - DTT dithiothreitol - 2-ME 2-mercaptoethanol - PVDF polyvinylidene difluoride The authors thanks P. Carrier and Dr. B. Dimon for technical assistance with the mass-spectrometry measurements.