Coated-vesicle formation in vitro: conflicting results using different assays

Cell-free systems provide essential tools for elucidating the molecular mechanisms underlying complex cellular processes such as vesicular transport. The biochemical utility of these model systems is strengthened by assays that allow rapid, quantitative detection of the events being studied. Two model systems have recently been developed to reconstitute coated-vesicle budding, and two different biochemical assays are used to detect this event. Striking differences in the biochemical requirements for 'coated-vesicle budding' are detected by these two assays, suggesting that two distinct events are being measured. These findings have wide implications for the use of cell-free assay systems in cell biology.     

Zolpidem Displays Heterogeneity in Its Binding to the Nonhuman Primate Benzodiazepine Receptor In Vivo

Abstract: The distinctive pharmacological activity of zolpidem in rats compared with classical benzodiazepines has been related to its differential affinity for benzodiazepine receptor (BZR) subtypes. By contrast, in nonhuman primates the pharmacological activity of zolpidem was found to be quite similar to that of classical BZR agonists. In an attempt to explain this discrepancy, we examined the ability of zolpidem to differentiate BZR subtypes in vivo in primate brain using positron emission tomography. The BZRs were specifically labeled with [¹¹C]flumazenil. Radiotracer displacement by zolpidem was monophasic in cerebellum and neocortex, with in vivo Hill coefficients close to 1. Conversely, displacement of [¹¹C]flumazenil was biphasic in hippocampus, amygdala, septum, insula, striatum, and pons, with Hill coefficients significantly smaller than 1, suggesting two different binding sites for zolpidem. In these cerebral regions, the half-maximal inhibitory doses for the high-affinity binding site were similar to those found in cerebellum and neocortex and ～100-fold higher for the low-affinity binding site. The low-affinity binding site accounted for <32% of the specific [¹¹C]-flumazenil binding. Such zolpidem binding characteristics contrast with those reported for rodents, where three different binding sites were found. Species differences in binding characteristics may explain why zolpidem has a distinctive pharmacological activity in rodents, whereas its pharmacological activity in primates is quite similar to that of classical BZR agonists, except for the absence of severe effects on memory functions, which may be due to the lack of substantial zolpidem affinity for a distinct BZR subtype in cerebral structures belonging to the limbic system.     

Testosterone-regulated expression of enzymes involved in steroid and aromatic hydrocarbon catabolism in Comamonas testosteroni.

The effect of testosterone as the sole carbon source on protein expression was analyzed in Comamonas testosteroni. Testosterone simultaneously induced the expression of steroid- and aromatic hydrocarbon-catabolizing enzymes and repressed one amino acid-degrading enzyme. It is suggested that steroids play a regulative role in catabolic enzyme synthesis during adaptive growth of C. testosteroni.     

Arginine modifiers as energy transfer inhibitors in photophosphorylation.

Photophosphorylation by spinach chloroplasts is inhibited after they have been incubated in the dark with either phenylglyoxal or butanedione. Inhibition by phenylglyoxal is strongest when N-ethylmorpholine is the buffer used during the incubation; that by butanedione requires the presence of borate as buffer. The inhibitions are not reversed by simply washing out the inhibitor, suggesting that a covalent modification of one or more arginine residues is responsible. This is supported by the reversibility of the butanedione inhibition if both the inhibitor and borate buffer are removed. ATPase of the chloroplasts, and of extracted protein, is inhibited, whether activated by trypsin or by heating. This indicates that arginine residues of the coupling factor are the probable major site(s) for attack by these modifiers, leading to the observed inhibitions.     

The ribosomal protein composition of the archaebacterium Sulfolobus

Summary Ribosomal subunits from the thermoacidophilic Archaebacterium Sulfolobus were purified and their protein composition analyzed by gel electrophoretic methods. A tentative nomenclature was proposed. 30S subunits contained 27, and 50S subunits 34, electrophoretically distinguishable proteins. Three additional proteins were present on both the 30S and 50S subunits. The protein pattern of three geographically different isolates of Sulfolobus (Italy, Japan, Yellowstone) were nearly identical.     

Relationships among raffinose plasmids determined by the immunochemical cross-reaction of their alpha-galactosidases.

Plasmid-encoded alpha-galactosidase served as a marker enzyme for the recognition and comparison of raffinose (Raf) plasmids present in strains of Escherichia coli. Immunochemical relationships were established among Raf plasmids of 39 independent isolates from man and domestic animals (from three continents) by using antiserum against alpha-galactosidase. Immunodiffusion revealed three serological subclasses of alpha-galactosidase, which are correlated with the biological and geographical origin of the host strains. It is concluded that the raf determinants of all Raf plasmids tested have evolved from a common ancestor.     

The rate of interconversion between the two unfolded forms of ribonuclease A does not depend on guanidinium chloride concentration.

The interconversion between the fast-folding and slow-folding forms of ribonuclease A is unaffected by the protein denaturant guanidinium chloride, between 2.8 m and 7.0 m, at 10 °C. Thus the rate of this reaction is insensitive to denaturants, in contrast to the model proposed by Kanehisa &; Tsong (1979). This result is consistent with other evidence that the interconversion reaction is proline isomerization.