Changes in the intracellular accumulation and distribution of metallothionein in rat liver and kidney during postnatal development

Metallothionein (MT) bound to zinc and copper was detected in high concentration in fetal and newborn rat livers by a cadmium saturation method. The levels of both hepatic zinc and MT remained high for the first 14 days after birth and decreased to adult levels by 24 days of age. There was a direct linear relationship between hepatic metallothionein and zinc concentrations during the first 31 days after birth. The ratio of MT to zinc levels also decreased with age suggesting a rapid degradation of MT during postnatal development. Immunohistochemical localization of MT by peroxidase-antiperoxidase technique, using a specific antibody to MT, showed intense intranuclear staining for MT in fetal and newborn rat liver which persisted until Day 9. The nuclear MT staining decreased with age; at 11 days it was equal both in nucleus and cytoplasm and at 14 days, MT was localized mainly in the cytoplasm, similar to adult rat liver pattern. The intranuclear localization of MT in neonates could be considered as a typical fetal-neonatal morphological pattern and its subsequent presence in the cytoplasm, an adult pattern.     

A new biochemical effect of erythropoietin

Erythropoietin stimulated lactoperoxidase (LP) or horseradishperoxidase (HRP) catalyzed iodination of tyrosine, the stimulation being more pronounced in case of LP. The diiodotyrosine (DIT) formation was stimulated more than MIT formation although the total quantity of DIT formed was less than MIT. Both sheep plasma erythropoietin and human urinary erythropoietin exhibited the above phenomenon and the stimulatory effects were directly propertional to their potencies. Preincubation of erythropoietin with neuraminidase abolished its stimulatory effect. Other glycoprotein hormones like thyrotropic hormone (TSH) and human chorionic gonadotrophin (HCG) did not stimulate the above system.     

Dielectric properties and oxygenation of hemoglobin

Dielectric properties of human and horse hemoglobin were studied at frequencies ranging from 20 kc./sec. to 7 Mc./sec. The relative errors in the measurements were usually less than 10^?3 even for mildly conducting solutions (10^?3M KCl). The experimental setup allowed us variation and measurement of the degree of oxygenation of the protein and to determine its dielectric parameters. Our main conclusion is that it was not possible to find any variation of the dielectric increment for hemoglobin oxygenation levels of 25, 50, 75, and 100%, approximately. This result is at variance with some previous reports. We cannot give the reason for this discrepancy but discuss some possible explanations. The specific dielectric increment, Δε_s/c, of human hemoglobin was shown to be significantly smaller than that of horse hemoglobin (0.28 against 0.32). This physical property is lowered with increasing ionic strength I: Δε_s/c = 0.28 and 0.20 for I = 10^?4 and 10^?3, respectively (human protein).     

Role of extracellular signaling on endothelial cell proliferation and protein N-glycosylation

In vitro studies of angiogenic phenomenon have been limited due to nonavailability of a simple and biologically relevant model of the capillary wall. Recent development of a capillary endothelial cell line from the vascular bed of bovine adrenal medulla made us to study the effect of heparin, thrombin, thyroxine, glucagon, insulin, and phorbol myristate acetate (PMA) on the proliferative and metabolic activities such as glycosylation of asparagine-linked glycoproteins of these cells in culture. Out of six different agents studied here, only heparin, thrombin, and thyroxine reduced the doubling time of these cells by 24 hr with no observed morphological abnormality. Glucagon, showed marginal reduction in the cell doubling time. By contrast, insulin and PMA enhanced the doubling time. Insulin treatment though induced the S phase of cell cycle but it blocked the cells entry into the G2 + M phase. PMA arrested the cells in G0/G1 phase. The cellular response to protein N-glycosylation is increased in the presence of thyroxine, insulin, and thrombin and the effect is dose dependent. Further analysis on SDS-PAGE indicated that glycosylation of 80-120 kDa and 43 kDa glycoprotein species are enhanced when these cells are treated with insulin and thrombin. Glycopeptide generated from these glycoproteins suggested that they all carry "high mannose" and "complex" type oligosaccharide chains attached to their protein core.     

Cloning and sequence analysis of the Escherichia coli metH gene encoding cobalamin-dependent methionine synthase and isolation of a tryptic fragment containing the cobalamin-binding domain

A gene encoding cobalamin-dependent methionine synthase (EC 2.1.1.13) has been isolated from a plasmid library of Escherichia coli K-12 DNA by complementation to methionine prototrophy in an E. coli strain lacking both cobalamin-dependent and -independent methionine synthase activities (RK4536:metE, metHH). Maxicell expression of a series of plasmids containing deletions in the metH structural gene was employed to map the position and orientation of the gene on the cloned DNA fragment. A 6.3-kilobase EcoRI-SalI fragment containing the gene was cloned into the sequencing vector pGEM3B for double-stranded DNA sequencing; the MetH coding region consists of 3372 nucleotides. The enzyme was purified from an overproducing strain of E. coli harboring the recombinant plasmid, in which the level of methionine synthase was elevated 30- to 40-fold over wild-type E. coli. Recombinant enzyme is a protein of 123,640 molecular weight and has a turnover number of 1,450 min-1 in the standard assay. These values are to be compared with previously reported values of 133,000 for the molecular weight and 1,240-1,560 min-1 for the turnover number of the homogenous enzyme purified from a wild-type strain of E. coli B (Frasca, V., Banerjee, R. V., Dunham, W. R., Sands, R. H., and Matthews, R. G. (1988) Biochemistry 27, 8458-8465). Limited proteolysis of the native enzyme with trypsin resulted in loss of enzyme activity but retention of bound cobalamin on a peptide fragment of 28,000 molecular weight. This fragment has been shown to extend from residue 643 to residue 900 of the 1124-residue deduced amino acid sequence.