Inactivation of gamma-aminobutyric acid aminotransferase by (Z)-4-amino-2-fluorobut-2-enoic acid

(Z)-4-Amino-2-fluorobut-2-enoic acid (1) is shown to be a mechanism-based inactivator of pig brain gamma-aminobutyric acid aminotransferase. Approximately 750 inactivator molecules are consumed prior to complete enzyme inactivation. Concurrent with enzyme inactivation is the release of 708 +/- 79 fluoride ions; transamination occurs 737 +/- 15 times per inactivation event. Inactivation of [3H]pyridoxal 5'-phosphate ([3H]PLP) reconstituted GABA aminotransferase by 1 followed by denaturation releases [3H]PMP with no radioactivity remaining attached to the protein. A similar experiment carried out with 4-amino-5-fluoropent-2-enoic acid [Silverman, R. B., Invergo, B. J., & Mathew, J. (1986) J. Med. Chem. 29, 1840-1846] as the inactivator produces no [3H]PMP; rather, another radioactive species is released. These results support an inactivation mechanism for 1 that involves normal catalytic isomerization followed by active site nucleophilic attack on the activated Michael acceptor. A general hypothesis for predicting the inactivation mechanism (Michael addition vs enamine addition) of GABA aminotransferase inactivators is proposed.     

Activation of 2-5A-dependent RNase by analogs of 2-5A (5'-O-triphosphoryladenylyl(2'----5')adenylyl(2'----5')adenosine ) using 2',5'-tetraadenylate (core)-cellulose

A variety of 2-5A (px(A2'p)nA; x = 2 or 3, n greater than or equal to 2) analogs were assayed for their abilities to activate murine 2-5A-dependent RNase (subsequently "the nuclease") using a recently developed method. This technique consists of immobilizing and partially purifying the nuclease using core-cellulose [A2'p)3A-cellulose) and then monitoring the breakdown of poly(U)-3'-[32P]Cp into acid-soluble fragments. Several 5'-adenosinecapped analogs of 2-5A (containing a tetra-, tri-, or diphosphate) were analyzed, and it was found that reducing the number of phosphoryl groups between the 5' to 5'-diadenosine linkages resulted in a progressive loss of activity. Because A5' pppp(A2'p)3A was a potent activator of the nuclease yet stable during the assay these results suggested that a free 5'-phosphoryl group may not be required for the activation of the nuclease. A number of 8-bromoadenosine-substituted analogs of 2-5A were also studied. Curiously, the brominations decreased the activities of the 5'-di- and triphosphorylated molecules while substantially increasing the activities of the 5'-monophosphorylated species. The results indicated that a tri- or diphosphate moiety on the 5'-end of 2-5A or the presence of ATP is not absolutely required for the nuclease to be active. Furthermore, the ATP analog, beta, gamma-methylene ATP, did not inhibit the activity of the nuclease. Finally, a 3',5'-phosphodiester linkage isomer of 2-5A and a 3'-deoxy (cordycepin) analog of 2-5A were tested, and both were found to be completely without activity.     

Movement of ions and small solutes across endothelium and epithelium of perfused rabbit lungs

In situ and isolated fluid-filled rabbit lungs were used to study the transport of indicators between the air space and vascular compartments. These indicators were placed in either the perfusate or air spaces and samples were collected from the perfusate at intervals during a 1-h perfusion period. At the end of the hour, fluid was pumped out of the air space compartment into serial tubes and indicator concentrations were determined in both the air space and perfusion fluids. One hour after introducing the indicators into the air space, the relative decreases in solute concentration were (arranged from the greatest to the least decline): [14C]urea greater than 36Cl- = 125I- greater than 22Na+ greater than [3H]mannitol. The relative rates at which the indicators appeared in the perfusate were similar. When the indicators were placed in the perfusate, a similar relationship was observed in the increase in air space concentrations, but the loss of 22Na+ from the perfusate was similar to those of 36Cl- and 125I-. Losses of all indicators from the perfusate were two or more times those from the air spaces, and although the loss of [3H]mannitol from the perfusate was similar to that of 22Na+ for about 30 min, subsequent loss was much slower. Very little 125I-albumin traversed the tissue barrier, and the small changes in the concentrations of 125I-albumin in the air spaces suggested that little fluid movement had occurred. These studies suggest that the epithelium is less permeable to solutes than the endothelium and permits passage of anions at a faster rate than 22Na+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of the hydration of CO2 catalyzed by carbonic anhydrase III from cat muscle

Using stopped flow methods, we have measured the steady state rate constants and the inhibition by N3- and I- of the hydration of CO2 catalyzed by carbonic anhydrase III from cat muscle. Also, using fluorescence quenching of the enzyme at 330 nm, we have measured the binding of the sulfonamide chlorzolamide to cat carbonic anhydrase III. Inhibition by the anions was uncompetitive at pH 6.0 and was mixed at higher values of pH. The inhibition constant of azide was independent of pH between 6.0 and 7.5 with a value of KIintercept = 2 X 10(-5) M; the binding constant of chlorzolamide to cat carbonic anhydrase III was also independent of pH in the range of 6.0 to 7.5 with a value Kdiss = 2 X 10(-6) M. Both of these values increased as pH increased above 8. There was a competition between chlorzolamide and the anions N-3 and OCN- for binding sites on cat carbonic anhydrase III. The pH profiles for the kinetic constants and the uncompetitive inhibition at pH 6.0 can be explained by an activity-controlling group in cat carbonic anhydrase III with a pKa less than 6. Moreover, the data suggest that like isozyme II, cat isozyme III is limited in rate by a step occurring outside the actual interconversion of CO2 and HCO3- and involving a change in bonding to hydrogen exchangeable with solvent water.     

Translocation of photoassimilate from leaves of two popyploid genotypes of tall fescue differing in photosynthetic rates

The photosynthetic rate of a decaploid genotype (1-16-2) of tall fescue ( Festuca arundinacea Schreb.) is about twice that of a common hexaploid genotype (V6-802) (Plant Physiol. 72: 16–21, 1983). Translocation of photosynthate out of the leaves is a possible means of regulating carbon assimilation. To evaluate this possibility, we have examined a) translocation velocity, b) time course of translocation from leaves, c) photoassimilate partitioning pattern into whole plants in pulse and chase experiments, and d) interveinal distances between two ploidy genotypes. Most of the ¹⁴C accumulated in sucrose, and the labelled carbon moved down the leaf blades at similar velocities (6 to 10 cm h⁻¹) in both genotypes. Recent ¹⁴C assimilate was rapidly translocated from the fed area of the leaf blade. For example, the decaploid and the common hexaploid had translocated 40 and 26% of the ¹⁴C, respectively, at 6 h, and 79 and 49% of the ¹⁴C, respectively, at 24 h. Partitioning of ¹⁴C among plant organs was considerably different between the genotypes after a 24 h chase. For example, out of the total ¹⁴C recovered from the whole plant, the decaploid had retained 40% in the labelled leaf with 10, 33 and 29% in other leaves, stem bases and roots, respectively; whereas the hexaploid had retained 91% in the labelled leaf with 4, 3 and 2% in other leaves, stem bases and roots, respectively. However, the higher rate of translocation was correlated with greater interveinal distances in the decaploid genotype. These results suggested that the higher translocation percentage in the decaploid than the hexaploid genotype was due to greater sink activity.     

Polymyalgia rheumatica/giant cell arteritis in a Cambridge general practice.

The aim of this study was to establish the incidence and prevalence of polymyalgia rheumatica/giant cell arteritis in general practice. Patients with this disorder, whether previously diagnosed or not, were ascertained by using a questionnaire administered by interview, and all received full clinical and laboratory assessment. A total of 579 patients aged 65 and over was seen, and 19 (33/1000) had been diagnosed or developed symptoms within the previous eight years. Thus the calculated annual incidence in those aged 65 and over was about 4/1000. The figures from this first large scale study of polymyalgia rheumatica/giant cell arteritis in general practice are much higher than those from studies carried out in hospital. The questionnaire was effective in both identifying known cases of polymyalgia rheumatica/giant cell arteritis and detecting new cases. As this is a treatable disorder, it is important that doctors become aware of how common it is in elderly people.     

Assay for characterization of human follicular oocyte maturation inhibitor using Xenopus oocytes

Human follicular fluid from healthy mature Graafian follicles and from pathologic ovarian cyst fluid was found to be inhibitory to progesterone-induced meiotic maturation of oocytes from the South African clawed toad, Xenopus laevis. Human follicular fluid but not human serum, collected from the same individuals, demonstrated a linear dose-response inhibition on the maturation of oocytes in the Xenopus assay system. These findings indicate that the human follicular and cyst fluids contain oocyte maturation inhibitor (OMI). This human OMI was inactivated when subjected to a boiling water bath for 2 min. The OMI action was shown to be reversible in its inhibitory action. The fact that OMI can act directly on the oocyte was demonstrated by its inhibitory action on maturation in defolliculated oocytes. The findings demonstrate that the inhibitory action of human OMI is not species-specific. Xenopus oocytes provide a simple, readily available, year-round bioassay material for testing follicular oocyte maturation inhibitor.     

In vitro and in vivo effects on brain GABA metabolism of (S)-4-amino-5-fluoropentanoic acid,a mechanism-based inactivator of γ-aminobutyric acid transaminase

The effects of intraperitoneal administration of (S)-4-amino-5-fluoropentanoic acid, a mechanism-based covalent inactivator of γ-aminobutyric acid transaminase (GABA-T), on whole brain GABA metabolism in mice were investigated. A dose-dependent and time-dependent irreversible inactivation of GABA-T was observed with a concomitant increase in whole brain GABA levels. The compound exhibited no $\text{in vitro}$ nor $\text{in vivo}$ time-dependent inhibition of glutamate decarboxylase (GAD), alanine transaminase, or aspartate transaminase (Asp-T). It was, however, a potent competitive reversible inhibitor of GAD and a weak competitive inhibitor of Asp-T. The chloro analogue, (S)-4-amino-5-chloropentanoic acid, was ineffective.     

Topographical distribution of the gonadotrophs,mammotrophs, somatotrophs and thyrotrophs in the pituitary gland of the baboon (Papio cynocephalus)

The identification and regionalization of four pituitary parenchymal cell types, gonadotrophs, mammotrophs, somatotrophs and thyrotrophs, were studied in the baboon (Papio cynocephalus) hypophysis using immunocytochemistry. The gonadotrophs were homogeneously distributed throughout the entire pars distalis. Both mammotrophs and somatotrophs predominate at the superior and inferior poles of the organ. The medial and anteromedial regions are populated by mammotrophs and thyrotrophs, while the lateral and posterior portions of the pars distalis contain large numbers of somatotrophs.