GABA and its related enzymes in the lower auditory system of the guinea pig

The possible existence of GABA-transmitter neurons in the lower auditory system of the guinea pig has been investigated by means of three different experimental approaches: (1) the regional distribution of GABA and its related enzymes, (2) the subcellular distribution of glutamate decarboxylase, and (3) the effect of selected nerve lesions on glutamate decarboxylase concentrations in the auditory nuclei. Within the regions investigated considerable variations in glutamate decarboxylase activity and GABA concentration were found, with the highest values observed in the inferior colliculus. The dorsal cochlear nucleus also contained significant amounts of both glutamate decarboxylase and GABA, in addition to high concentrations of GABA transaminase. The subcellular distribution of glutamate decarboxylase was bimodal in both the cochlear nucleus and inferior colliculus with most enzyme activity recovered in the soluble and synaptosomal fractions. Neither end organ (cochlea) nor trapezoid body lesions induced a significant loss of glutamate decarboxylase activity in either the cochlear nucleus or inferior colliculus. The results suggest the presence of short axon GABAergic interneurons in the cochlear nucleus, most of which appear to terminate within the dorsal cochlear nucleus.     

Quantitative Isolation of Undegraded Polysomes from Aged Pea Tissue in the Absence of Contaminants and Artefacts

Undegraded polysomes were isolated successfully from aged peaepicotyls by grinding frozen tissue in at least 10 volumes ofbuffer A (0.2 M Tris-HCl, pH 8.5; 0.2 M sucrose; 60 mM KCl;30 mM MgCl₂), taking care to prevent the tissue from thawingprior to homogenization. Supposedly pure polysomes, derivedfrom the membrane-bound polysome fraction, were apparently contaminatedwith membranes, and contained polysomes clumped together vianascent chains. Problems with contaminants and artefacts werepartially alleviated by the use of polyoxyethylene tridecylether as a detergent replacing Triton X-100; further alleviatedby the use of large volumes of detergent-containing buffer toresuspend the membrane-bound polysome; and almost completelyeliminated by brief treatment of resuspended polysomes withprotease K. Optimal conditions for isolating polysomes fromaged tissue are given. ¹ Present address: Institute of Agricultural Environment Control,College of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama790, Japan. (Received April 24, 1985; Accepted September 2, 1985)     

Broad-range and binary-range acyl-acyl-carrier protein thioesterases suggest an alternative mechanism for medium-chain production in seeds.

In the current model of medium-chain (C8-14) fatty acid biosynthesis in seeds, specialized FatB acyl-acyl-carrier-protein (ACP) thioesterases are responsible for the production of medium chains. We have isolated and characterized FatB cDNAs from the maturing seeds of elm (Ulmus americana) and nutmeg (Myristica fragrans), which accumulate predominantly caprate (10:0)- and myristate (14:0)-containing oils, respectively. In neither species were we able to find cDNAs encoding enzymes specialized for these chain lengths. Nutmeg FatB hydrolyses C14-18 substrates in vitro and expression in Brassica napus seeds leads to an oil enriched in C14-18 saturates. Elm FatB1 displays a binary specificity: one activity is centered on 10:0-ACP, and a second is centered on palmitate (16:0)-ACP. After expression in B. napus seeds the oil is enriched in C10-18 saturates, predominantly 16:0, 14:0, and 10:0. The composition of free fatty acids produced by elm FatB1 in Escherichia coli shifts from C14-16 to mostly C8-10 by increasing the rate of chain termination by this enzyme. These results suggest the existence of an alternative mechanism used in the evolution of medium-chain production, a model of which is presented.     

Conformational changes in the extracellular β-lactamase I from Bacillus cereus 569/H/9

1. The thermal denaturation and precipitation of beta-lactamase I from Bacillus cereus 569/H/9 at 60 degrees C are reversible, a soluble and almost fully active enzyme being obtained after solution of the precipitate in 5m-guanidinium chloride or 8m-urea and subsequent removal of the denaturing agent. 2. Inactivation of beta-lactamase I occurs rapidly between 50 degrees and 55 degrees C and is shown by circular-dichroism spectra to be accompanied by an extensive conformational change. 3. A change to a different conformation occurs in 6m-urea. This change is also reversible; refolding with almost complete recovery of enzymic activity occurs within 5min of dilution of the denaturing agent. 4. Inactivation of beta-lactamase I at pH3.0 and 11.0 is also associated with conformational changes, since a proportion of the lost activity is recovered within 5min of adjustment of the pH to 7.0.