Biomedical subjects

M A Snoswell

Publications and source records attributed to M A Snoswell.

5 recordsLinked to original sources

PubMed1992

Isolation of a carboxyphosphate intermediate and the locus of acetyl-CoA action in the pyruvate carboxylase reaction.

When chicken liver pyruvate carboxylase was incubated with either H14CO3- or gamma-[32P]ATP, a labeled carboxyphospho-enzyme intermediate could be isolated. The complex was catalytically competent, as determined by its subsequent ability to transfer either 14CO2 to pyruvate or 32P to ADP. While the carboxyphospho-enzyme complex was inherently unstable and the stoichiometry of the transfer was variable depending on experimental conditions, both the [14C]carboxyphospho-enzyme and the carboxy[32P]phospho-enzyme had similar half-lives. Acetyl-CoA was shown to be involved in the conversion of the carboxyphospho-enzyme complex to the more stable carboxybiotin-enzyme species, which was consistent with the effects of acetyl-CoA on isotope exchange reactions involving ATP. We were unable to detect the formation of a phosphorylated biotin derivative during the ATP cleavage reaction. In the presence of K+ and at pH 9.5, the acetyl-CoA-independent activity of chicken liver pyruvate carboxylase approached 2% of the acetyl-CoA-stimulated rate, which represents a 30-fold increase on previously reported activity for this enzyme.

Acetyl Coenzyme A↗

PubMed1985

Complete nucleotide sequence of hepatic 5-aminolaevulinate synthase precursor.

Chick embryo liver mitochondrial matrix protein, 5-aminolaevulinate synthase, is synthesised initially as a larger cytosolic precursor. In this report we present the complete nucleotide sequence of a cDNA clone coding for the precursor together with corresponding confirmatory amino acid sequence of peptides derived from purified mature mitochondrial enzyme. The deduced amino acid sequence shows that the precursor consists of mature enzyme of 579 amino acids and an N-terminal extension of 56 amino acids. The latter presequence is highly basic in character as found with other mitochondrial preproteins.

5-Aminolevulinate Synthetase↗

PubMed1985

Pyruvate carboxylase: mechanisms of the partial reactions.

Data from isotopic exchange studies and from experiments with 32P- and 14C-labeled enzyme-bound intermediates support the following description of the first partial reaction: (Formula: see text). From studies of the transfer of the carboxyl-group from ENZ-biotin-CO2- to pyruvate or its analogues we propose that binding of the acceptor substrate induces the translation of carboxybiotin from the first to the second partial reaction site. The studies on the translocation of carboxybiotin can be summarized in the following reaction scheme: (Formula: see text). Where k+3 less than k+1, k-1, k+2 and k-2. Thus, the rate-limited step is governed by k+3 which represents the movement of carboxybiotin from the first subsite to the second.

Acetyl Coenzyme A↗

PubMedDate not supplied

Sizing biological samples by photosedimentation techniques.

The performance of the Joyce-Loebl disk centrifuge in the sizing of Escherichia coli cells, protein inclusion bodies, and cell debris is evaluated. The need for a density gradient that extends throughout the entire spin fluid is highlighted, and a set of standard conditions that fulfill this requirement is defined. E. coli cells experience a reduction in their Stokes diameter when exposed to ethanol, indicating that a spin-buffer fluid combination such as glycerol-water is to be preferred for the sizing of bacteria. The instrument baseline is influenced by the presence of particles, and a method of estimating the baseline is described. The sizing of small particles is further complicated by baseline drift due to temperature sensitivity of the optical yoke. An analysis of diffusion in the spin fluid is conducted, and an expression for the sedimentation:diffusive flux ratio is derived. For the current samples, it is shown that diffusion within the spin fluid does not lead to significant errors for 0.15-microns particles, whereas the phenomenon may be significant at the manufacturer's size limit of 0.01 micron.

Centrifugation, Density Gradient↗

PubMedDate not supplied

High-resolution particle size analysis in biotechnology process control.

Many industrially important proteins can now be expressed intracellularly as insoluble protein inclusion bodies. In production, large-scale centrifugation is commonly used to separate and recover the inclusion bodies. Recovery efficiency depends critically on the centrifuge feed rate, which must be optimized to minimize production costs. We have used a disc centrifuge photosedimentometer to make high-resolution measurements of the particle size distribution (PSD) of the supernatant during the production of porcine somatotropin (pST) inclusion bodies. These measurements readily monitor the breakthrough of inclusion bodies into the supernatant and allow the centrifugation operation to be optimized.

Biotechnology↗