Expression of rat renal gamma-glutamyltransferase cDNA in Escherichia coli

To obtain the expression of rat kidney gamma-glutamyltransferase (GGT) cDNA in E. coli, plasmids containing the cDNA sequences coding for various parts of GGT were constructed. Transformation of E. coli cells by these hybrid vectors results in a production of unglycosylated recombinant proteins, immunologically recognized by specific antirat kidney GGT antibodies. Plasmid, expressing the complete coding sequence of GGT cDNA, allows the production of enzymatically active proteins localized in the periplasmic space, while the same sequence without the N-terminal hydrophobic region results in a production of cytoplasmic proteins. These recombinant proteins present a very basic isoelectric point (pI greater than 9). These results suggest that the presence of the N-terminal region seems to be necessary to direct the expressed proteins enzymatically active in the periplasmic space.     

Production,cross reactivity,and epitope analysis of monoclonal antibodies against rat kidney gamma glutamyltransferase

Eighteen IgGl monoclonal antibodies (blabs) have been produced against gamma-glutamyl transferase (GGT) from rat kidney. They were specific to the light subunit of the enzyme with affinity constants ranging from 0.3 to 7.5 10⁸ M^–1, while they did not react with GGT from other sources i.e. human and pig kidney, rat and guinea pig liver, suggesting species and organ specificity. Two of the blabs (N° 11 and 21) lost their immunoreactivities towards rat kidney GGT in the presence of N-acetyl-neuraminic acid, while immunoreactivities of the other blabs were unchanged. Furthermore, Mabs No 11 and 21 did not react with desialylated rat kidney GGT. These findings suggest that N-acetyl-neuraminic acid is involved in the epitopes recognized by these two Mabs.Abbreviations ELISA enzyme linked immunosorbent assay - GGT gamma-glutamyltransferase - Mab monoclonal antibody - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Coordinated changes in enzyme activities of phenylpropanoid metabolism during the growth of soybean cell suspension cultures

Variations in teh activities of several enzymes of phenylpropanoid metabolism were studied in fermenter-grown cell suspension cultures of soyben (Glycine max).Concomitant large increases and subsequent decreases in the activities of phenylalanine ammonina-lyase (EC 4.3.1.5), cinnamic acid 4-hydroxylase, and two isoenzymes of p-coumarate:CoA ligase occurred prior to the stationary phase of the cell cultures. These findings represent a further example of an interdependent regulation of these enzymes of the general phenylpropanoid metabolism.The increases in all of these enzyme activities could be further enhanced by illunination of the cells.No comparable light effects and no significant changes were observed for the specific activity of an S-adenosylmethionine:o-dihydric phenol m-O-mehyltransferase and for the overall rate of the two-step reduction of feruloyl-CoA to coniferyl alcohol. These enzymatic reactions therefore appear to be regulated independently of the enzymes of the general phenylpropanoid metabolism.     

Effects of decreased atmospheric deposition on the sulfur budgets of two Dutch moorland pools

The chemical composition of surface waters of two Dutch moorland pools and of incident precipitation, was monitored from 1982 to 1990. For this period, sulfur and water budgets were calculated using a hydrochemical model developed for well-mixed non-stratifying lakes. Total atmospheric deposition of S decreased significantly after 1986 at both locations. A model describing the sulfur budget in terms of input, output and reduction/oxidation processes predicted a fast decrease of pool water SO₄ ²⁻ concentrations after a decrease of atmospheric input. However, SO₄ ²⁻ concentrations in the surface water was lowered only slightly or remained constant. Apparently a source within the lake caused the unexpectedly high SO₄ ²⁻ concentrations. The possible supply of SO₄ ²⁻ from the sediment through regulation by (K-)Al-SO₄ containing minerals or desorption of SO₄ ²⁻ from positively charged surfaces in the sediment was evaluated. Solubility calculations of pore water with respect to alunite, basaluminite and jurbanite indicated that SO₄ ²⁻ concentration was not regulated by these minerals. It is suggested here (1) that desorption of SO₄ ²⁻ from peaty sediments may account for the estimated SO₄ ²⁻ supply provided that the adsorption complex is periodically recharged by partial oxidation of the upper bottom sediments and (2) that because of exposure of a part of the pool bottom to the atmosphere during dry summers and subsequent oxidation of reduced S, the amount of SO₄ ²⁻ may be provided which complements the decreasing depositional SO₄ ²⁻ input. In future research these two mechanisms need to be investigated.     

Sulfate reduction and S-oxidation in a moorland pool sediment

In an oligotrophic moorland pool in The Netherlands, S cycling near the sediment/water boundary was investigated by measuring (1) SO₄ ^2– reduction rates in the sediment, (2) depletion of SO₄ ^2– in the overlying water column and (3) release of³⁵S from the sediment into the water column. Two locations differing in sediment type (highly organic and sandy) were compared, with respect to reduction rates and depletion of SO₄ ^2– in the overlying water.Sulfate reduction rates in sediments of an oligotrophic moorland pool were estimated by diagenetic modelling and whole core³⁵SO₄ ^2– injection. Rates of SO₄ ^2– consumption in the overlying water were estimated by changes in SO₄ ^2– concentration over time in in situ enclosures. Reduction rates ranged from 0.27–11.2 mmol m^–2 d^–1. Rates of SO₄ ^2– uptake from the enclosed water column varied from –0.5, –0.3 mmol m^–2 d^–1 (November) to 0.43–1.81 mmol m^–2 d^–1 (July, August and April). Maximum rates of oxidation to SO₄ ^2– in July 1990 estimated by combination of SO₄ ^2– reduction rates and rates of in situ SO₄ ^2– uptake in the enclosed water column were 10.3 and 10.5 mmol m^–2 d^–1 at an organic rich and at a sandy site respectively.Experiments with³⁵S^2– and³⁵SO₄ ^2– tracer suggested (1) a rapid formation of organically bound S from dissimilatory reduced SO₄ ^2– and (2) the presence of mainly non SO₄ ^2–-S derived from reduced S transported from the sediment into the overlying water. A³⁵S^2– tracer experiment showed that about 7% of³⁵S^2– injected at 1 cm depth in a sediment core was recovered in the overlying water column.Sulfate reduction rates in sediments with higher volumetric mass fraction of organic matter did not significantly differ from those in sediments with a lower mass fraction of organic matter.Corresponding author     

Footprint of the sigma protein: a re-examination

Escherichia coli RNA Polymerase is a multi-subunit enzyme that catalyzes RNA synthesis, using DNA as a template. The sigma subunit of this enzyme plays an important role in the recognition of promoter sites on DNA. Using DNase I footprinting, Utpala Ramesh and Claude F. Meares [(1989) Biochem. Biophys. Res. Comm. 160, 121-125] reported that in the absence of the other subunits, sigma binds specifically to the bacteriophage lambda PR promoter DNA sequence. We are unable to reproduce that result.