Comparative study of phosphopeptides in various animal tissues]

We determined the phosphopeptide (PP) level in several tissues and compared the values to the phosphoprotein and phosphatide level. We studied whole organs and subcellular fractions isolated from these organs. The results showed that the PP were preferentially localised in membranes. Among all the organs analyzed, the electrical organ of "Torpedo marmorata" had the highest PP level. The phosphoprotein distribution was different from the PP one; the highest level of phosphorproteins was found in nuclei. There was also an increase in phosphoprotein level comparing whole tissues and membranes isolated from the same tissue. The preferential localisation of PP in membranes is also suggested by the parallelism between PP and phosphatide level, which is also higher in membranes than in whole organs. The peculiar polyanionic structure, as well as the active metabolism and the membranous localisation of PP support the hypothesis that these compounds might be good intermediates in active transport mechanisms.     

Comparative structural and enzymatic properties of skeletal muscle myosin from neonatal and adult rabbits

Several structural and enzymatic properties of myosin from skeletal muscles of neonatal and adult rabbits were compared. Electrophoretic analyses and proteolysis experiments indicated that differences between the two myosin types could be attributed to their heavy subunits. Circular dichroism measurements of subfragment-1 species, and trypsin-digested derivatives showed that the neonatal protein contained less alpha-helices than the adult form. The Mg2(+)-ATPase activity of neonatal myosin was lower than that of adult myosin, especially in the presence of actin. In comparison with adult subfragment-1, it was found that the binding of ATP analogues such as adenosine 5'-[beta, gamma-imino]triphosphate and PPi, or that of ATP (as deduced from the apparent KmATP) to neonatal subfragment-1 in the presence of actin was enhanced, while that of ADP was decreased. On the other hand, the association of actin with the ADP - neonatal-subfragment-1 complex was weaker. These features must be expressed in the cyclical actin-myosin association/dissociation steps occurring in ATP hydrolysis, and more particularly in the reassociation of actin with the ATP-hydrolysis-products - myosin complex.     

Soluble endothelin degradation enzyme activities in various rat tissues.

From soluble extract of rat kidney we have previously identified an endothelin degradation enzyme that rapidly and specifically cleaves off the C-terminal tryptophan of endothelin-1, resulting in a peptide that is three orders of magnitude weaker in potency than endothelin-1 in causing smooth muscle contraction. The tissue distribution of this enzyme was examined, and the soluble extracts of rat kidney were found to contain the highest enzyme activity, followed by the spleen and the liver. In contrast, no enzyme activity was detected in the soluble extracts of brain, heart, and lung. The biochemical properties of the partially purified enzyme from kidney were further investigated. The optimal pH of the enzyme was between 5 and 7. The endothelin degrading activity was effectively blocked by thiol protease inhibitors such as benzyloxycarbonyl-Phe-Ala-diazomethyl ketone and p-hydroxymercuribenzoic acid, as well as by phenylmethylsulfonyl fluoride, but not by metalloprotease and other serine protease inhibitors. This enzyme displayed a clear difference in substrate specificity when compared with other thiol proteases such as cathepsin B, cathepsin H, and cathepsin L, known to be present in the kidney. These results suggest that a novel protease with endothelin degrading activity is widely distributed in a number of tissues.     

Catalase enzymatic activity and electrophoretic patterns in adult amphibians--a comparative study

Catalase electrophoretic patterns and enzymatic activities were measured in four organs of two anuran species, Rana ridibunda perezii and Discoglossus pictus. The D. pictus enzyme appeared as two distinguishable bands, whereas R. ridibunda catalase was monomorphic. Electrophoretic mobility of the major D. pictus catalase band was greater than that of R. ridibunda. Enzymes from both species showed slower mobility than that from bovine liver. Catalase activities did not show significant differences according to sex in any of the organs tested in R. ridibunda. Enzyme activities were similar in liver, kidney and brain when both species were compared. Only the heart showed much higher activity in D. pictus than in R. ridibunda. The catalase activity levels followed the order: liver greater than kidney greater than heart in both species. The heart showed higher activity than the brain in D. pictus but not in R. ridibunda.     

6-phosphogluconolactonase. Purification, properties and activities in various tissues

6-Phosphogluconolactonase was purified to apparent homogeneity in a four-step procedure from bovine erythrocytes. The extent of purification and the kinetic properties of the enzyme were evaluated with an optical test that was based on the hydrolysis of synthetic 6-phosphogluconolactone. The active enzyme from bovine erythrocytes is a monomer with an approximate molecular weight of 30000. It exhibits Michaelis-Menten kinetics and cofactors are not required for activity. The enzyme was found in a number of tissues. Its activity, when compared to the activity of the glycolytic enzymes, illustrates the importance of glucose oxidation via the pentose phosphate pathway relative to glycolysis.     

Expression of transforming growth factor-beta 1 in specific cells and tissues of adult and neonatal mice

We have used immunohistochemical techniques to detect transforming growth factor-beta 1 (TGF-beta 1) in many tissues of adult and neonatal mice. Each of two antibodies raised to the amino-terminal 30 amino acids of TGF-beta 1 selectively stained this molecule in either intracellular or extracellular locations. Strong intracellular staining was found in adrenal cortex, megakaryocytes and other cells of the bone marrow, cardiac myocytes, chondrocytes, renal distal tubules, ovarian glandular cells, and chorionic cells of the placenta. Marked staining of extracellular matrix was found in cartilage, heart, pancreas, placenta, skin, and uterus. Staining was often particularly intense in specialized cells of a given tissue, suggesting unique roles for TGF- beta within that tissue. Levels of expression of mRNA for TGF-beta 1 and its histochemical staining did not necessarily correlate in a given tissue, as in the spleen. The present data lend further support to the concept that TGF-beta has an important role in controlling interactions between epithelia and surrounding mesenchyme.     

Suppressor activity in the spleen of neonatal mice.

Helper cells of T-cell origin are required for the in vitro proliferation of low numbers of adult mouse thymus cells in response to allogeneic spleen cells. These helper cells are present in the adult mouse spleen. We have demonstrated that neonatal mouse spleen cells lack the helper activity present in adult spleen cells. We have also shown that this lack of helper activity is the result of active suppression. The suppression is due to a suppressor cell which is present in high quantities in the neonatal spleen and can be eliminated by treatment with anti-θ serum and complement.