Biomedica biochimica acta最新文献

The two major tissue forms of N-acetyl-beta-D-glucosaminidase, A and B, and a minor form M were isolated from human urine by chromatography on DEAE-Trisacryl M and Con A-Sepharose. Forms A and B have a Km-value of about 0.75 mM for 4-nitrophenyl-beta-2-acetamido-2-deoxy-D-glucopyranoside, form M a corresponding value of 1.57 mM. The pH optimum was for all forms between pH 4.0 and 4.8. Forms A and M were heat labile, while form B was relatively heat stable. Using different binding properties of forms A and B on Trisacryl, a batch method was developed to differentiate both forms in human urine. This method can be easily performed and gives reproducible results. The intra-run and between-run precision for the separation of the two forms were below 10%.

In human, goat and chick amniotic fluid the contents of lecithin, lysolecithin and phosphatidyl ethanolamine increase during embryonic development. The sphingomyelin content however, increased during the early period only and declined during the latter part of development.

Liver and intestinal nucleotides and energy charge levels were examined in rats at portal vein clamping for 1, 5 or 30 min and after reflow for 1 or 6 h following 30 min clamping. In both tissues, clamping resulted in a loss of ATP, energy charge, UTP, GTP, UDP-glucuronic acid and UDP-N-acetylhexosamine levels, which all, except ATP and total adenosine phosphates, essentially recovered at 1 h reflow. At 6 h reflow, liver UDP-glucuronic acid and liver glutathione decreased in both clamped and sham operated rats.

Recombinant desulfatohirudin variant 1 is efficiently expressed and secreted from Saccharomyces cerevisiae. Chemical analysis of the secreted hirudin compounds revealed the presence of the full-length hirudin molecule as well as two degradation products that lack the C-terminal and in addition the penultimate amino acid, respectively. To eliminate the yeast proteases possibly involved in C-terminal hirudin proteolysis, we disrupted either the structural gene for endoprotease yscA (PRA1) or the gene encoding carboxypeptidase yscY (PRC1). Both isogenic mutant strains secreted significantly higher amounts of full-length hirudin as compared to the parental strain. This suggests an involvement of carboxypeptidase yscY in hirudin proteolysis, since both protease disruptions lead to a lack in yscY activity; a yscA mutant accumulates the inactive yscY precursor. However, the strain devoid of protease yscA yielded significantly lower titers of total hirudin than the strain lacking yscY, but containing yscA.

Daily administration of the trapidil derivative AR 12463 (20 mg/kg body weight i.p.) to cholesterol-fed rabbits diminished statistically significantly the increase in serum total cholesterol. After 8 weeks of treatment all measured lipoprotein fractions were significantly lower in animals treated with AR 12463 (group 3) compared with the values of the untreated control (group 1) or vehicle-treated group (group 2). The reduction of serum levels was associated with statistically significantly reduced levels of cholesterol esters in kidney, liver and aorta. The levels of free cholesterol in the liver of group 3 animals were statistically significantly lower compared with the levels in the two other groups, whereas in kidney and aortas the levels of free cholesterol remained unchanged under the influence of AR 12463. The area of aorta covered with fatty streaks was significantly smaller in group 3 versus group 1. The results of this study indicate that treatment of rabbits with AR 12463 while feeding a cholesterol-rich diet prevents hypercholesterolemia as well as the cholesterol incorporation into tissues. The mode of action of AR 12463 on serum and tissue lipids as well as on the development of atherosclerosis is discussed.

Aqueous two-phase systems containing polyethylene glycol (PEG) and dextran as phase forming polymers were used for the partition of unmodified and hypochlorite modified lipoproteins. Low density lipoprotein (LDL) was separated from high density lipoprotein (HDL) by sequential ultracentrifugation from human plasma. In agreement with the higher electrophoretic mobility, high density lipoprotein shows a higher value of the partition coefficient in contrast to low density lipoprotein. An increase in the concentration of chloride ions reduces the enrichment of lipoprotein in the top phase and favours the accumulation of aggregated material at the interface. The partition coefficient strongly depends on the age of the lipoprotein sample. Differences in the value of the partition coefficient could be obtained for the lipoprotein fractions HDL-2 and HDL-3. Hypochlorite modified LDL shows higher values of the partition coefficient due to the higher negative charge of the modified lipoprotein particle.

The in vitro transfer of radiolabelled unesterified cholesterol from human low- and very low- to high-density lipoproteins in the presence of either lipoprotein deficient serum or bovine serum albumin has been studied. The rate of transfer was faster from LDL (t1/2 = 44 min) than from VLDL (55 min). The presence of 2% protein had no effect on the transfer. However, 10% albumin or lipoprotein deficient serum reduced t1/2 by 35%, which indicates no specific effect of a plasma protein on the rate of transfer of unesterified cholesterol.

A peptidase from the non pathogenic Staphylococcus sp. strain BEC 299 was purified to a final specific activity of 84,400 U/mg protein. Its molecular weight is 450 kDa and optimum pH 10.0. This enzyme catalyzes the synthesis of dipeptides (aspartame) and alpha-amino acid derivatives (N-L-malyl-L-tyrosine ethyl ester). The influence of cosolvents and pH on dipeptides and alpha-amino acid derivative synthesis is described. Finally, we detail the use of the peptidase as a reagent in protease-catalyzed peptide synthesis.