Quinuclidine chemistry: autocondensation reactions of 3-quinuclidinone

During the synthesis of 3-hydroxy-3-ethinylquinuclidine (I), two additional products were isolated and identified as (E)-3-[2-(3-oxoquinuclidine)]quinuclidylidene (III) and (E)-3-[2-(3-hydroxy-3-ethinylquinuclidine)]quinuclidylidene (V). The base-catalyzed autocondensation of 3-quinuclidinone resulted in the alpha,beta-unsaturated ketone dimer (III) as a single isomer. The geometric configuration was deduced by examination of the NMR spectra of the methyl iodide salt. Compound V was thus the result of attack on the carbonyl carbon of III by the acetylide anion. The isolation and identification of these compounds clarified the reported differences in the physical properties of I and its analogs.     

The subunit structure of bovine brain 14-3-2

Two aspects of the subunit structure of the bovine brain specific protein 14-3-2 have been examined. On the one hand, native 14-3-2 has been separated into two fractions by hydroxylapatite chromatography. One eluted at the same position when chromatographed on the same column, while the other redistributed into the same two fractions again. Amino acid analysis of these two forms of 14-3-2 gave results that were not significantly different under the condition of analysis. Furthermore, when each peak was subjected to cyanogen bromide cleavage, very similar elution profiles of the resultant fragments were obtained. The two pools also cross-reacted with antiserum to 14-3-2. Reaction of purified 14-3-2 with dimethylsuberimidate caused the formation of covalently bound protein units of 100,000 molecular weight when measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis, as opposed to the 50,000 minimal molecular weight normally detected. On the other hand, analysis of the soluble tryptic peptides of S-[14C]carboxymethyl 14-3-2 yielded only three distinct radioactive peptides, each with one residue of S-carboxymethylcysteine whereas 8 are expected on the basis of the amino acid composition of the 50,000 molecular weight polypeptide chains. Thermolysin digestion of a similarly modified 14-3-2 preparation yielded all of the radioactivity in 5 S-carboxymethylcysteine-containing peptides. The partial amino acid sequence of these peptides indicates that they represent 4 unique areas of the polypeptide chain. Since 8 such peptides were expected, that is, double the number found, the minimum structural unit of the protein must be of 25,000 molecular weight. The results of these experiments do not permit distinction between a duplication of the structure within a single polypeptide chain or the alternate possibility of two polypeptide chains bound by unusually strong non-covalent bonds. These results suggest that 14-3-2 is a covalently linked dimer of 25,000 mol.wt. units that can aggregate to form larger species of 100,000 mol.wt. and higher.     

The serum kinetics of bovine testicular hyaluronidase in dogs, rats and humans

There is experimental and clinical evidence that i.v. injection of bovine testicular hyaluronidase (BTH) reduces the extent of necrosis during myocardial infarction. The fate of i.v. administered BTH has not been described. In this study, serum kinetics of BTH enzyme activity in dogs, rats and humans were determined. Tissue distribution of BTH was determined with an 125I-labeled preparation of purified BTH. Serum BTH activity initially decreased exponentially with half-life 2.0 +/- 0.1 min in dogs with coronary artery occlusion (n = 8; 500 U of BTH/kg); 3.2 min in humans with acute myocardial infarction (n = 2; 500 U of BTH/kg); and 3.2 +/- 0.3 min in rats (n = 5; 5,000 U of BTH/kg). In dogs BTH disappearance showed two distinct phases. After injection of high dose BTH (5,000 U of BTH/kg), during the first 7 min serum half-life of BTH was 2.1 +/- 0.2 min (n = 8), but increased to 9.4 min in later serum samples. After the injection of 125I-labeled BTH into the rat, protein-bound 125I disappeared from serum with a half-life (3.4 min) that is similar to the serum half-life of BTH enzyme activity (3.2 min). Twenty minutes after injection of 125I-labeled BTH, 30% of the label was recovered in the liver. It is concluded that BTH activity has a short serum half-life of less than 10 min in dogs, rats and humans. In the rat model, the disappearance of serum BTH activity results from physical removal of circulating BTH molecules rather than serum inhibition or inactivation of BTH enzymatic activity.     

Estimation of serum gentamicin by quenching fluoroimmunoassay

A new type of non-isotopic immunoassay, applied to the determination of serum gentamicin, is reported. The method is based on partial quenching of fluorescence observed when fluorescein-labelled gentamicin is bound by anti-gentamicin serum. The fluorescence intensity of the labelled gentamicin in an unseparated immunoassay incubation mixture therefore serves to indicate the extent of binding, which is related to the amount of competing unlabelled gentamicin present. Precision and accuracy are shown to be similar to those of the best existing methods for gentamicin, while the new assay is more rapid and technically simpler, and avoids the use of expensive radio-chemicals with their attendant health hazard. Assays of patient samples correlate with established bioassay and polarisation fluoroimmunoassay methods.