Thin-film antimony-antimony-oxide enzyme electrode for penicillin determination

A potentiometric penicillinase electrode is reported in which the base pH transducer is a thin-film anti-mony-antimony-oxide electrode deposited by vacuum evaporation. Several enzyme immobilization procedures have been examined and a crosslinked protein film found to be the most appropriate to this type of sensor. The use of an adjacent antimony-antimony-oxide track as a pseudoreference electrode was successfully demonstrated. The overall response was shown to be independent of the stirring rate above 100 rpm, but the kinetics of the response were found to depend markedly on the stirring rate. The intrinsic linear response range was 3 x 10(-4)Mto 7 x 10(-3)M penicillin G. Linearizing transforms that extend the useful range were examined.     

Nucleotide sequence and evolution of the orangutan ε globin gene region and surrounding Alu repeats

Summary We have mapped and sequenced the globin gene and seven surrounding Alu repeat sequences in the orangutan globin gene cluster and have compared these and other orangutan sequences to orthologously related human sequences. Noncoding flanking and intron sequences, synonymous sites of , , and globin coding regions, and Alu sequences in human and orangutan diverge by 3.2%, 2.7%, and 3.7%, respectively. These values compare to 3.6% from DNA hybridizations and 3.4% from the globin gene region. If as suggested by fossil evidence and molecular clock calculations, human and orangutan lineages diverged about 10–15 MYA, the rate of noncoding DNA evolution in the two species is 1.0–1.5×10^–9 substitutions per site per year. We found no evidence for either the addition or deletion of Alu sequences from the globin gene cluster nor is there any evidence for recent concerted evolution among the Alu sequences examined. Both phylogenetic and phenetic distance analyses suggest that Alu sequences within the and globin gene clusters arose close to the time of simian and prosimian primate divergence (about 50–60 MYA). We conclude that Alu sequences have been evolving at the rate typical of noncoding DNA for the majority of primate history.Presented at the FEBS Symposium on Genome Organization and Evolution, held in Crete, Greece, September 1–5, 1986     

Calcium-dependent anion channel in the water mold,Blastocladiella emersonii

Summary Injection of depolarizing current into vegetative cells of the water moldBlastocladiella emersonii elicits a regenerative response that has the electrical characteristics of an action potential. Once they have been taken past a threshold of about –40 mV, cells abruptly depolarize to +20 mV or above; after an interval ranging from several hundred milliseconds to a few seconds, the cells spontaneously return to their resting potential near –100 mV. When the action potential was analyzed with voltage-clamp recording, it proved to be biphasic. The initial phase reflects an influx of calcium ions through voltage-sensitive channels that also carry Sr²⁺ ions. The delayed, and more extended, phase of inward current results from the efflux of chloride and other anions. The anion channels are broadly selective, passing chloride, nitrate, phosphate, acetate, succinate and even PIPES. The anion channels open in response to the entry of calcium ions, but do not recognize Sr²⁺. Calcium channels, anion channels and calcium-specific receptors that link the two channels appear to form an ensemble whose physiological function is not known. Action potentials rarely occur spontaneously but can be elicited by osmotic downshock, suggesting that the ion channels may be involved in the regulation of turgor.     

The lipoxygenases in developing soybean seeds, their characterization and synthesis in vitro

A number of lipoxygenase isoenzymes were identified in developing soybean (Glycine max L. Merrill cv Provar) seeds and two have been partially characterized. In a study of lipoxygenase level in developing soybean seeds, the enzyme content increased markedly during development. Comparisons of the lipoxygenases from mature soybean seeds and immature seeds by isoelectric focusing, chromatofocusing, sodium dodecyl sulfate polyacrylamide gel electrophoresis and peptide mapping identified two categories of isoenzyme. The isoenzymes from immature seeds were found by electron paramagnetic resonance spectroscopy to be isolated at least in part as the high spin iron(III) or active form of the enzyme in contrast to lipoxygenases from mature seeds which were isolated as electron paramagnetic resonance silent, high spin iron(II) species. The discovery of increased levels of lipoxygenases during seed development and their isolation in an active form suggests that the enzyme may play a physiological role during the maturation process. The incorporation of iron-59 from the nutrient medium into lipoxygenase during culture of immature seeds was indicative of de novo synthesis of the enzyme. The efficiency of the iron uptake was high, as indicated by the level of radioactivity found in the enzyme (one gram atom of iron per mole of lipoxygenase).     

Ionophore-induced efflux of sodium and potassium ions from sea urchin eggs

The efflux of K⁺ and Na⁺ from sea urchin eggs during Ca²⁺ ionophore A23187-induced parthenogenesis was studied in a K⁺ and Na⁺-free artificial seawater using extracellular ion-specific electrodes. We have probed this model system with monovalent cation-specific ionophores to determine if they affect K⁺ efflux in the unfertilized egg and whether any changes in ionophore sensitivity are observed during egg activation. In 500 mM choline chloride, 10 mM CaCl₂, 50 mM MgCl₂, 10 mM Tris-Cl pH 8.0, A23187 induced a rapid efflux of K⁺ and Na⁺ from the eggs after a short lag time (10–15 seconds). After the burst, the rate of K⁺ efflux remained higher than the pre-activation rate, but was lower than during the burst phase, while the rate of Na⁺ efflux became nearly zero. Monovalent cation-specific ionophores (valinomycin, gramicidin and nigericin) had no effect on K⁺ efflux from the unfertilized eggs in our model system. However, once the egg was activated by A23187, each of the above ionophores caused a prolongation of the burst phase for many minutes. These results show that the unfertilized egg plasma membrane (using our artificial conditions) is not susceptible to the monovalent cation-specific antibiotics and suggest that either the inserted cortical granule membrane or the developing fertilization envelope interacts with these ionophores to cause the change in rate-limiting step for K⁺ efflux observed egg activation.     

Transposon donor plasmids, based on ColIb-P9, for use in Pseudomonas putida and a variety of other gram negative bacteria

Summary The properties of pLG221, a derivative of the ColIb plasmid carrying the transposon Tn5 are described. This plasmid can be used to introduce Tn5 by conjugation from Escherichia coli into a variety of Gram negative bacteria outside the host range for maintenance of ColIb. Plasmid pLG221, and a similar plasmid pLG223 carrying Tn10 may be of general utility as vectors for transposon-mediated mutagenesis in a variety of Gram negative bacteria.     

A Supernodulation and Nitrate-Tolerant Symbiotic (nts) Soybean Mutant

The nodulation characteristics of soybean (Glycine max) mutant nts382 are described. The mutant nodulated significantly more than the parent cultivar Bragg in the presence and absence of several combined nitrogen sources (KNO₃, urea, NH₄Cl, and NH₄NO₃). The number of nodules on the tap root and on lateral roots was increased in the mutant line. In the presence of KNO₃ and urea, nitrogenase activity was considerably higher in nts382 than in Bragg. Mutant plants were generally smaller than wild-type plants. Although nts382 is a supernodulator, inoculation with Rhizobium japonicum was necessary to induce nodule formation and both trial strains CB1809 (= USDA136) and USDA110 elicited the mutant phenotype. Segregation of M₃ progeny derived from a M₂ wild-type plant indicated that the mutant character is inherited as a Mendelian recessive. The mutant is discussed in the context of regulation of nodulation and of hypotheses that have been proposed to explain nitrate inhibition of nodulation.     

The topography of glycerophosphate acyltransferase in the transverse plane of the mitochondrial outer membrane

In low ionic media, mitochondrial glycerophosphate acyltransferase was inhibited virtually completely within 15 min by the nonspecific proteases, proteinase K and subtilisin. In high ionic media, the mitochondrial enzyme was either not inhibited or was marginally inhibited by these proteases. Chymotrypsin and trypsin, regardless of the ionic strength of the medium, did not inhibit the acyltransferase. Substantial inhibition by proteinase K and subtilisin was observed in the high ionic media when the incubation was continued for 30 or 45 min. Adenylate kinase, an intermembrane enzyme, was not inhibited under any of the above conditions. These results demonstrate a cytosolic exposure of the mitochondrial acyltransferase. In a low ionic environment, when the outer membrane integrity was damaged either by gradually decreasing the tonicity of the medium or by stepwise addition of Triton X-100, either chymotrypsin or trypsin caused virtually parallel inhibition of glycerophosphate acyltransferase and adenylate kinase. A more direct approach in establishing the existence of protease-susceptible sites on the inner side of the outer membrane was taken by observing the inhibition of mitochondrial glycerophosphate acyltransferase and adenylate kinase in trypsinloaded right-side-out outer membrane vesicles incubated in the presence of externally located soybean trypsin inhibitor. The above results, taken together, suggest that mitochondrial glycerophosphate acyltransferase spans the transverse plane of the outer membrane.     

Comparison of Three 18F-Labeled Butyrophenone Neuroleptic Drugs in the Baboon Using Positron Emission Tomography

The butyrophenone neuroleptics spiroperidol, benperidol, and haloperidol were radiolabeled with fluorine-18 and studied in baboon brain using positron emission transaxial tomography (PETT). Pretreatment of the baboon with a high pharmacological dose of (+)-butaclamol reduced the specifically bound component of radioactivity distribution in the striatum to approximately the radioactivity distribution found in the cerebellum. Comparative studies of brain distribution kinetics over a 4-h period indicated that either [18F]spiroperidol or [18F]benperidol may be suitable for specific labeling of neuroleptic receptors. In an 8-h study with [18F]spiroperidol, striatal radioactivity did not decline, suggesting that spiroperidol either has a very slow dissociation rate or that it binds irreversibly to these receptors in vivo. [18F]Haloperidol may not be suitable for in vivo PETT studies, because of a relatively high component of nonspecific distribution and a faster dissociation from the receptor. Analysis of 18F in plasma after injection of [18F]spiroperidol indicated rapid metabolism to polar and acidic metabolites, with only 40% of the total radioactivity being present as unchanged drug after 30 min. Analysis of the metabolic stability of the radioactively labeled compound in rat striatum indicated that greater than 95% of [18F]spiroperidol remains unchanged after 4 h.