Protein extraction by reverse micelles: a study of the factors affecting the forward and backward transfer of alpha-chymotrypsin and its activity

alpha-chymotrypsin is taken as a model protein to investigate three aspects of the protein extraction by reverse micelles: (1) the comparison between the two forward transfer techniques, i.e., the liquid-liquid and the solid state-liquid transfer; (2)the back-transfer, i.e., the capability of the protein to be recovered from the micellar solution; and (3) the maintainance of the enzyme activity at the end of the extraction cycle. Concerning the forward transfer from the liquid phase, we study first the effect of salt initially present in the aqueous phase on the equilibrium concentration of the extracted species; further, we study the forward protein extraction from the solid state, and the effect of pH, salt, and protein concentration on the transfer efficiency. Concerning the back transfer, we find the somewhat surprising result, that the percentage of protein back-extraction depends on the type and concentration of salt used for the forward transfer. Preliminary data concerning an alternative method for the back-transfer using silica gel to liberate the protein from the micellar environment, are presented. Finally, it is found that the enzyme activity depends again on the type and concentration of salt used for the forward transfer.     

An improved approach for transformation of plant cells by microinjection: molecular and genetic analysis

A new culture method for the injection of tobacco mesophyll protoplasts has been established. The protoplasts are embedded in a thin layer of alginate and are nourished from the medium in the underlying basislayer. In the alginate layer the protoplasts regenerate to calli at a frequency of up to 80%. Embedded protoplasts can be selected either with 50 mg l⁻¹ kanamycin or 5 mg l⁻¹ paromomycin. Single resistant cells can be recovered from about 10 000 sensitive cells in one alginate layer. Injection of theneo gene (coding for neomycin phosphotransferase II) into protoplast derived single cells in the alginate layer results in kanamycin resistant colonies that can be regenerated to mature plants. These plants express the neomycin phosphotransferase as shown by enzyme activity assay. The integration of the transgene into the plant genome could be proved by Southern hybridization to high molecular weight DNA. With this culture method 100 cells can be injected per hour. Transformation frequencies range from 2 to 20%. In crossing experiments, it was shown that the foreign gene is transmitted to the next generation in a Mendelian fashion.     

Spectroscopic and binding studies of azide to type-2-copper-depleted ascorbate oxidase from zucchini

Summary Binding of azide to type-2-copper-depleted (T2D) zucchini ascorbate oxidase, containing reduced type-3 Cu centers, and met-T2D ascorbate oxidase, containing oxidized type-3 Cu centers, has been studied spectroscopically. In both cases titration with azide in 0.1 M phosphate pH 6.8 produces a broad near-ultraviolet band with maximum at 455 nm (e 2500 M^–1 cm^–1, with respect to the met-T2D enzyme) and shoulder at 390 nm (e 1700 M^–1 cm^–1), that are assigned to(azide)Cu(II) ligand-to-metal charge transfer (LMCT) transitions. This is accompanied by a reduction of absorbance at 330 nm in the met-T2D) enzyme adduct (e –1400 M^–1 cm^–1). A broad circular dichroic band of negative sign between 370–480 nm corresponds to the LMCT absorption band. Analysis of the titration data indicates that one azide ion binds independently to each of the binuclear T3 Cu couples with low affinity (K = 50 M^–1). The ESR signal of the T1 Cu observed in frozen solutions of the T2D enzyme is also perturbed by the addition of azide. The analogies in the azide-binding characteristics between ascorbate oxidase and laccase are discussed.     

Steroid modulation of oxytocin/vasopressin receptors in the uterus

Oxytocin (OT) and V1 vasopressin (VP) receptors are present simultaneously in several tissues, including the uterus. In myometrium these receptors mediate contractility, while in endometrium they mediate the release of other uterotonic substances as endothelin (ET). In rabbit myometrium, estrogens increase, while progesterone blunts neurohypophysial hormone receptors. However, the action of sex steroids on OT and V1 VP receptors differs in terms of the ED₅₀ and maximal effect. Therefore, at parturition, only OT receptors show a dramatic rise, while V1 VP receptors do not change, suggesting a major role for OT in labor. ET is a potent stimulator of uterine activity acting through specific receptors present on myometrial cells. These receptors as well as the endometrial localization of ET are modulated by sex steroids, indicating that ET might represent a paracrine regulator of uterine activity. In humans, OT but not V1 VP receptors increase as pregnancy progresses, confirming the primary relevance of OT in timing delivery.     

MK-801 Prevents 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Parkinsonism in Primates

In cynomologus monkeys, systemic administration of MK-801, a noncompetitive antagonist for the N-methyl-D-aspartate receptor, prevented the development of the parkinsonian syndrome induced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MK-801 also attenuated dopamine depletion in the caudate and putamen and protected dopaminergic neurons in the substantia nigra from the degeneration induced by the neurotoxin. Nevertheless, 7 days after MPTP administration in the caudate and putamen of monkeys also receiving MK-801, the levels of toxic 1-methyl-4-phenylpyridinium were even higher than those measured in monkeys receiving MPTP alone. This indicates that the protective action of MK-801 is not related to MPTP metabolism and strongly suggests that, in primates, the excitatory amino acids could play a crucial role in the mechanism of the selective neuronal death induced by MPTP.     

Chirality of reverse micelles

Four chiral analogues of the surfactant Aerosol-OT (AOT) have been synthesized and characterized. All of them form reverse micelles in apolar solvents in the w₀ range 0–30 (w₀ = [water]/[tenside]). Reverse micellar solutions have been investigated by UV absorption and circular dichroism spectroscopies with the aim of clarifying whether the formation of the macromolecular micellar structure induces the appearance of new chromophoric bands or perturbs the existing ones. Methanolic solutions of the surfactants, in which no micellar aggregates are formed, were taken as references. One of the products 1(S),1′(S)-dimethylbisheptylsulphosuccinate sodium salt (MH-AOT) was capable of forming reverse micelles of relatively high water content (w₀ up to 40) and this process was accompanied by a specific increase in the intensity of the circular dichroism band associated with the ester absorbance of the molecule. As no concomitant changes were seen in the UV absorbance spectrum, it was concluded that this observation reflected conformational events occurring within the surfactant rather than chromophoric perturbation. These results are qualitatively similar to those found recently for lecithin reverse micelles which, however, form gels at sufficiently high water contents. The chiroptical properties of these supramolecular aggregates are compared with those of covalent macromolecular systems such as polypeptides.     

Effect of Kainic Acid, Glutamate, and Aspartate on CO2 Production by Goldfish Tectal Slices

For a study of the excitatory effect of kainate, glutamate, and aspartate in the goldfish optic tectum, these substances were tested on the production of CO2 from radioactive glucose in tectal slices incubated in Krebs-Ringer medium for fish. Kainate increased the rate of CO2 production for up to 30 min in a dose-related manner, the effect being maximum at 0.1 mM concentration and decreasing at higher doses. The effect was blocked by ouabain (1 mM) as well as by the substitution of choline for Na+ in the incubation medium. Glutamate and aspartate exerted a less pronounced excitatory effect on CO2 production at higher concentration than kainate. This effect was also abolished by ouabain. Glutamate, added to the medium at a concentration at least 100-fold higher than kainate, partially reversed the increase in CO2 production induced by kainic acid. No similar effect was noticed for aspartate. The supposed glutamate antagonists glutamic acid diethylester (1 mM) and proline (5 mM) did not affect the excitatory action of kainic acid or exert an antagonistic effect towards glutamate. At higher concentration (10 mM) glutamic acid diethylester increased CO2 production, an effect that was, however, ouabain insensitive. Methyltetrahydrofolic acid (1 mM), a substance reported to compete for the kainate receptor, did not inhibit the effect of kainic acid or increase CO2 production.     

Location and characterization of opiate receptors regulating pituitary secretion

The site at which opiate agonists and antagonists act to alter secretion of prolactin, growth hormone and luteinizing hormone as well as the pharmacological specificity of the opiate receptors mediating these effects were examined in rats. Injection of β-endorphin but not a 10 fold higher dose of the non opiate peptide β-endorphin, increased release of prolactin and growth hormone in male rats while inhibiting luteinizing hormone release in ovariectomized, estrogen primed female rats. Prior treatment with naltrexone i.p. blocked these responses. Injection of naltrexone into the hypothalamus lowered prolactin release. In rats with a surgically formed hypothalamic island systemic administration of morphine or naltrexone altered prolactin release in the same manner as was observed in intact animals. In contrast no effects of β-endorphin or naltrexone were observed on the spontaneous secretion of prolactin $\text{in}$$\text{vitro}$. In addition β-endorphin did not alter the inhibition of prolactin release produced by apomorphine $\text{in}$$\text{vitro}$. The ED₅₀ for stimulation of prolactin release following intraventricular administration of β-endorphin or the synthetic enkephalin analog FK 33-824 was the same, approximately 0.1 ng/rat. However FK 33-824 at 0.2 ng/rat was able to produce much greater analgesia and catatonia than β-endorphin. The metabolism and distribution of β-endorphin was examined but did not account for these differential effects. These results indicate that opiate agonists and antagonists can act at the hypothalamic but not the anterior pituitary level to alter release of prolactin, growth hormone and luteinizing hormone. In addition the data suggest that the opiate receptors mediating release of prolactin may have a different pharmacological specificity from those involved with analgesia and catatonia.     

Co-oligopeptides containing two aromatic residues spaced by glycyl residues. X. Proton magnetic resonance study of co-oligopeptides of tryptophan and glycine

The ¹H-nmr spectra of co-oligopeptides of tryptophan and glycine with structure H-Gly-Trp-(Gly)_n-Trp-Gly-OH (n = 0–2) and those of several di- and tripeptides have been recorded at 360 MHz with CD₃OD solutions containing 0.1N NaOD. The assignment of resonance signals was generally possible by comparing the spectra of structurally related peptides with each other. In order to solve the remaining ambiguities in the assignment, H-(αL,βS)(α,β-d₂)Trp-OH, H-Trp-(αL,βS)(α,β-d₂)Trp-OH, and H-Trp-(δ¹,ε²,ζ²,ζ³,η²-d₅)Trp-OH have been prepared and their spectra compared with those of the undeuterated compounds. The distribution of rotamers around the χ₁ and (in two cases) χ₂ torsion angles of the side chains has been obtained from the vicinal coupling constants ³J and from the long-range coupling constants ⁴J. These data and an analysis of the chemical shifts of the Gly-C^α protons suggest that the orientation of the aromatic side chain is influenced by the following order of decreasing interaction with the functional groups at N- and C-side: -NH₂ > –NHCO– > –CONH–> –COO^?. This rule does not hold for the second Trp residue of di- and tripeptides containing the -Trp-Trp- sequence, which has tentatively been attributed to steric effects.     

Effects of diffusible products of peroxidation of rat liver microsomal lipids

The effects on cellular structures of products of peroxidation of rat liver microsomal lipids were investigated. A system containing actively peroxidizing liver microsomal fraction was separated from a revealing or target system by a dialysis membrane. The target system, contained in the dialysis tube, consisted of either intact cells (erythrocytes) or subcellular fractions (liver microsomal fraction). When liver microsomal fractions were incubated with NADPH (or an NADPH-generating system), lipid peroxidation, as measured by the amount of malonaldehyde formed, occurred very rapidly. The malon-aldehyde concentration tended to equilibrate across the dialysis membrane. When the target system consisted of erythrocytes, haemolysis occurred abruptly after a lag phase. The lysis was greatly accelerated when erythrocytes from vitamin E-deficient rats were used, but no haemolysis was observed when erythrocytes from vitamin E-treated rats were used. When, in the same system, freshly prepared liver microsomal fractions were exposed to diffusible factors produced by lipid peroxidation, the glucose 6-phosphatase activity markedly decreased. A similar decrease in glucose 6-phosphatase activity, as well as a smaller but significant decrease in cytochrome P-450, was observed when the target microsomal fractions were exposed to diffusible factors derived from the peroxidation of liver microsomal lipids in a separate preincubation step. These and additional experiments indicated that the toxicological activity is relatively stable. Experiments in which the hepatic microsomal fractions destined for lipid peroxidation contained radioactively labelled arachidonic acid, previously incorporated into the membranes, showed that part of the radioactivity released from the microsomal fraction into the incubation medium entered the dialysis tube and was recovered bound to the constituents of the microsomal fractions of the target system. These results indicate that during the course of the peroxidation of liver microsomal lipids toxic products are formed that are able to induce pathological effects at distant loci.