Characterization of the glyceraldehyde 3-phosphate dehydrogenase from the extremely halophilic archaebacterium Haloarcula vallismortis.

Glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from the extremely halophilic archaebacterium Haloarcula vallismortis has been purified in a four step procedure to electrophoretic homogeneity. The enzyme is a tetramer with a relative molecular mass of 160,000. It is strictly NAD(+)-dependent and exhibits its highest activity in 2 mol/l KCl at 45 degrees C. Amino acid analysis and isoelectric focusing indicate an excess of acidic amino acids. Two parts of the primary sequence are reported. These peptides have been compared with glyceraldehyde 3-phosphate dehydrogenases from other archaebacteria, eubacteria and eucaryotes. The peptides show a high grade of similarity to glyceraldehyde 3-phosphate dehydrogenase from eucaryotes.

Partial purification and characterization of a growth factor from human hyperplastic prostatic tissues.

A growth factor capable of stimulating DNA synthesis of Balb/c 3T3 cells was purified by heparin-Sepharose column chromatography about 1900-fold from the cytosol of human prostatic tissues obtained at autopsy or open prostatectomy. This growth factor bound to heparin-Sepharose in the presence of 0.5 mol/l NaCl and was eluted by 1.0-1.55 mol/l NaCl. Its molecular weight was estimated to be 68,000 by SDS-polyacrylamide gel electrophoresis. The amino acid composition was determined and compared with the data of other growth factors, which revealed no striking conformity. Distribution of growth factor activity was investigated in mechanically separated prostatic tissues of benign prostatic hyperplasia. The separation scheme provided two fractions: the stromal fraction consisting mainly of fibroblasts, fibers and smooth muscle, and the epithelial fraction consisting of epithelial cells. The specific growth-stimulating activity in the stromal fraction was about 2-fold that in the epithelial fraction. Referred to the total activity of whole tissue, about 74% of the activity could be detected in the stromal fraction, while only about 5% was detectable in the epithelial fraction. This study demonstrates the existence of a growth factor in human benign hyperplastic prostatic tissues, showing a remarkable distribution of growth factor activity, which may play a role in the pathogenesis of benign prostatic hyperplasia.

Arginine deiminase from Halobacterium salinarium. Purification and properties.

Arginine deiminase from the extreme halophilic archaebacterium Halobacterium salinarium was purified to homogeneity in a four-step procedure with a 310-fold enrichment. The enzyme consists of two identical subunits of 55 kDa; its native molecular mass is 105 kDa. The pI of 4.7 indicates that acidic nature of the protein, which is evidenced by its amino acid composition, which shows an excess of more than 15% of acidic amino acids. The N-terminal amino acid of the enzyme is lysine. Arginine deiminase from Halobacterium salinarium exhibits its highest catalytic activity in the presence of 3.5 M-NaCl, pH 7.6, and at 40 degrees C. The half-activity constant, Ks, for arginine is 3.1 mM. The enzyme is inhibited by ornithine.

Purification and properties of alanine dehydrogenase from Halobacterium salinarium.

1. L-Alanine dehydrogenase (L-alanine:NAD+ oxidoreductase (deaminating), EC 1.4.1.1) was purified about 500-fold from Halobacterium salinarium. 2. The enzyme appears to be homogeneous in polyacrylamide gel electrophoresis. The apparent molecular weight is about 60 000. 3. Activity and stability of the enzyme are largely affected by different salts. Full activity of the NADH-dependent reductive amination of pyruvate occurs at 4.3 M NaCl. This activation can be achieved also by KCl and several other salts instead of NaCl. 4. The NAD+-dependent oxidative deamination of L-alanine occurs only in the presence of high concentrations of KCl. This reaction is not stimulated by NaCl. The Km values for the substrates NADH, pyruvate and NH+4 are also salt dependent. 5. The thermal stability of the enzyme is considerably higher in the presence of high concentrations of NaCl than in the presence of KCl. 6. The enzyme is completely inactivated by the removal of salt. Full reactivation is achieved by addition of salt in the presence of 2-mercaptoethanol. Inactivation proceeds about ten times faster than reactivation. The inactivation after the withdrawal of salt and the reactivation following the readdition of salt show a characteristic hysteresis loop.

Uptake and metabolism of long chain fatty acids in isolated chicken intestinal epithelial cells.

The uptake and metabolism of long chain fatty acids in isolated mucosal cells from chicken small intestine are studied. The viability of the isolated enterocytes is proven by linear oxygen consumption, CO2 and lactate formation form glucose and the active transport of glucose. The transport of palmitic and oleic acid is mediated by passive diffusion. This is demonstrated by the following results: (1) no saturation kinetics in the concentration range of 0.1--10.0 mM; (2) no competitive inhibition of the uptake by structurally related compounds; (3) no influence of 2,4-DNP and cyanide of the uptake; (4) the uptake is independent of sodium ions. Uptake rates of palmitic and oleic acid from suspensions are significantly higher than from the corresponding fatty acid-bovine serum albumin complexes. In both cases the uptake of palmitic acid proceeds faster than the uptake of oleic acid. Palmitic acid is oxidized to CO2 and incorporated into glycerides by enterocytes. Glucose serves as a glyceride-glycerol precursor. Its addition decreases the oxidation of the fatty acids and enhances glyceride synthesis markedly. Free glycerol is phosphorylated by enterocytes and can also serve as a glyceride-glycerol precursor.

Control of Extracellular beta-1,3-glucanase activity in a basidiomycete species.

The basidiomycete QM 806 excreted large amounts of beta-1,3-glucanase into the culture medium. Synthesis and excretion of the enzyme were triggered by a critically low concentration of carbon source. The extracellular beta-1,3-glucanase exhibited a remarkable stability. Addition of glucose or other carbon sources to a culture after consumption of the initial carbon source led to an inactivation of the extracellular beta-1,3-glucanase by an inactivating system, which could be separated from the cells. The inactivation of beta-1,3-glucanse was prevented by cycloheximide. This indicates the necessity of active protein synthesis for the inactivation process but does not prove that the inactivating system itself is a protein. Marked changes in the electrophoretic mobility and immunological properties of beta-1,3-glucanase indicate rather profound alterations of the enzyme protein in the course of inactivation.