Binding of manganese in human and rat plasma

Albumin, transferrin and 'transmanganin' have all been proposed as the major Mn-binding ligand in plasma. The present investigations were initiated in order to resolve these discrepancies. Compared to other metals tested (109 Cd2+, 65Zn2+, 59Fe3+), 54Mn2+ bound poorly to purified albumin. The addition of exogenous albumin to plasma did not result in an increased 54Mn radioactivity associated with this protein. Also, incubation of 65Zn-albumin in the presence of excess Mn2+ (1 mM) did not result in the displacement of Zn from albumin or Mn binding. In contrast to these results, 54Mn was bound to purified transferrin, not as readily as Fe3+, but better than Zn2+ or Cd2+. Saturation of transferrin with Fe3+ (1.6 micrograms Fe/mg) prevented the binding of 54Mn indicating that Mn probably binds to Fe-binding sites on the protein. Polyacrylamide gel electrophoresis further demonstrated the association of 54Mn with transferrin rather than with albumin in both human and rat plasma. The amount of 54Mn radioactivity recovered with transferrin increased as incubation time was increased, probably due to oxidation of Mn2+ to Mn3+. Mn binding to transferrin reached a maximum within 5 and 12 h of incubation. About 50% of 54Mn migrated with transferrin, whereas only 5% was associated with albumin. A significant portion (20-55%) of the 54Mn radioactivity migrated with electrophoretically slow plasma components whose identity was not determined. Possibilities include alpha 2-macroglobulin, heavy gamma-globulins and/or heavy lipoproteins.     

THE SECRETORY PATHWAYS OF RAT SERUM GLYCOPROTEINS AND ALBUMIN : Localization of Newly Formed Proteins within the Endoplasmic Reticulum

These studies compare the secretory pathways of newly formed rat serum glycoproteins and albumin by studying their submicrosomal localization at early times after the beginning of their synthesis and also by determining the submicrosomal site of incorporation of N-acetylglucosamine, mannose, galactose, and leucine into protein. N-acetylglucosamine, mannose, and galactose were only incorporated in vitro into proteins from membrane-attached polysomes and not into proteins from free polysomes. Mannose incorporation occurred in the rough endoplasmic reticulum, was stimulated by puromycin but not by cycloheximide, and 90% of the mannose-labeled protein was bound to the membranes. Galactose incorporation, by contrast, occurred in the smooth microsome fraction and 89% of the radioactive protein was in the cisternae. Albumin was mostly recovered (98%) in the cisternae, with negligible amounts in the membranes. To determine whether the radio-active sugars were being incorporated into serum proteins or into membrane protein, the solubilized in vivo-labeled proteins were treated with specific antisera to rat serum proteins or to albumin. Immunoelectrophoresis of the ¹⁴C-labeled leucine membrane and cisternal proteins showed that the membranes contained radioactive serum glycoprotein but no albumin, while the cisternal fraction contained all of the radioactive albumin and some glycoproteins. The results indicate that newly formed serum glycoproteins remain attached to the membranes of the rough endoplasmic reticulum after they are released from the membrane-attached polysomes, while albumin passes directly into the cisternae.     

Regulation of the ontogeny of rat liver metallothionein mRNA by zinc

To investigate the role of metals in the regulation of the ontogenic expression of rat liver metallothionein (MT) mRNA, the concentrations of zinc, MT and MT mRNA were determined in livers of fetal and newborn rats from dams which were fed with a control or zinc-deficient or copper-deficient or iron-deficient diet from day 12 of gestation. The liver samples were analyzed for MT-mRNA levels using a mouse MT-I cRNA probe. Although the newborn hepatic levels of each metal (zinc or copper or iron) was specifically reduced corresponding to the respective mineral deficiencies, the hepatic concentrations of total MT and MT-I mRNA were significantly decreased only in pups born from zinc-deficient dams. Injection of the zinc-deficient newborn pups with 20 mg Zn as ZnSO4/kg restored with MT-I mRNA levels to slightly above control values within 5 h of injection. The hepatic zinc, MT and MT-I mRNA levels were observed to increase significantly in control fetal rat liver on days 17-21 of gestation but there were little changes in either zinc or MT in fetal livers from zinc-deficient dams during the late gestational period. The MT-I mRNA level also did not show an increase on days 18 and 20 of gestation in zinc-deficient fetal liver as compared to controls. These results demonstrate a direct role of zinc in hepatic MT gene expression in rat liver during late gestation. Immunohistochemical localization of MT using a specific antibody to rat liver MT showed that the staining for MT in zinc-deficient pup liver was mainly in the cytosol in contrast to the significant nuclear MT staining observed in control newborn rat liver. The results suggest that maternal zinc deficiency has a marked effect not only in decreasing the levels of hepatic MT and MT-I mRNA but also in the localization of MT in newborn rat liver.     

T-lymphocyte heterogeneity in the rat: Separation of distinct rat T-lymphocyte populations which respond in syngeneic and allogeneic mixed lymphocyte reactions

These experiments were designed to determine if separate subpopulations of T cells were involved in the syngeneic and allogeneic mixed lymphocyte reaction. Rat lymph node T cells were separated into W3/25⁺ and W3/25^? subpopulations by panning with the monoclonal antibody W3/25 and tested for their ability to proliferate in both syngeneic (SMLR) and allogeneic (MLR) mixed lymphocyte responses, as well as to develop cytotoxicity against allogeneic, syngeneic, and trinitrophenol (TNP)-modified syngeneic targets. The W3/25⁺ T cells reacted strongly in the SMLR and the MLR whereas the W3/25^? fraction proliferated only in response to allogeneic stimulation and with a kinetic pattern distinct from W3/25⁺. Furthermore, addition of W3/25 monoclonal antibody directly to the cultures was shown only to inhibit the proliferation of the W3/25⁺ T-cell fraction. The W3/25^? subpopulation contained cytotoxic T cells (CTLs) against both allogeneic determinants and TNP-modified self. However the requirements for the activation of allospecific CTLs were distinct from those for CTLs for TNP-self in that W3/25^? allospecific CTLs required no detectable help from W3/25⁺ T cells but generation of the CTL response against TNP-self required the presence of W3/25⁺ helper T cells (Th). These data suggest that in the rat, there exist subsets of T cells recognized by their cell surface phenotype that distinguish between self and nonself determinants and the requirements for activation are different for each of these populations.     

A spectroscopic study of rat liver and Scylla serrata crab metallothioneins

The absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of native rat liver and crab (Scylla serrata) Cd,Zn-metallothionein have been measured and the data are compared. The MCD data indicate that there are close similarities in the geometries of the cadmium-binding sites in both of these proteins; however, the CD spectra are quite different for the rat liver and crab proteins. The CD spectrum for the crab metallothionein is unlike any previously reported for a cadmium-containing metallothionein. This suggests that the CD spectrum is sensitive to the different bridging pattern used in the binding sites in the crab compared with the rat-liver metallothionein. Cadmium binding to the metal-free metallothionein is demonstrated for both proteins and it is shown that there are only minor structural differences between the native and remetallated proteins. The structural changes that occur near to the cadmium-binding sites during cadmium loading to the native proteins have been followed using absorption and CD spectroscopy. Marked changes are observed in the CD spectrum which can be associated with a two-phase reaction: initially Zn2+ is displaced by the Cd2+, then at higher concentrations of Cd2+ the tetrahedral geometry of the Cd2+-binding sites is lost as more Cd2+ is bound using the same thiolate groups. While this latter reaction results in considerable change to the CD spectrum, only minor changes are observed in the absorption spectrum. A significant red shift is observed in the S leads to Cd charge transfer transition region of the MCD spectrum (230-270 nm) following both cadmium loading of native rat liver, Cd,Zn-metallothionein and the metallation of metal-free metallothionein with cadmium. There are two contributions to this effect in Cd,Zn-metallothionein: (i) there is a S leads to Zn band underlying the S leads to Cd band; and (ii) the occupation of zinc sites by cadmium changes the energy of the S leads to Cd transition.     

A heterogeneous beta-D-glucosidase activator from monkey parotid gland and its use as an affinity ligand in the purification of human salivary beta-D-glucosidase

We have isolated a heat-stable, low molecular weight activator peptide(s) from monkey parotid gland that specifically activated human salivary beta-D-glucosidase. This activator appeared to be heterogeneous on Sephadex G-25 gel filtration and polyacrylamide gel electrophoresis under non-denaturing conditions. About 45% of the human salivary beta-glucosidase could bind to the activator immobilised on Sepharose and be eluted by Cutscum. The purified enzyme was nearly homogeneous, with a subunit Mr of 46,000 as revealed by SDS-gel electrophoresis and silver staining.