Endogenous ADP-ribosylation of eukaryotic elongation factor 2 and its 32 kDa tryptic fragment.

Eukaryotic elongation factor 2 (eEF-2) can undergo ADP-ribosylation in the absence of diphtheria toxin. The binding of free ADP-ribose and endogenous transferase-dependent ADP-ribosylation were distinct reactions for eEF-2, as indicated by different findings. Incubation of eEF-2 tryptic fragment 32/33 kDa (32F) with NAD was ADP-ribosylated and gave rise to the covalent binding of ADP-ribose to eEF-2. 32F was revealed to be at the C-terminal by Edman degradation sequence analysis. In our study, the elution of 32F from SDS-PAGE was ADP-ribosylated both in the presence and absence of diphtheria toxin. These results suggest that endogenous ADP-ribosylation of 32F might be related to protein synthesis. This modification appears to be important for the cell function.

A quantitative colorimetric method of measuring alkaline phosphatase activity in eukaryotic cell membranes.

Alkaline phosphatase (ALP) is glycoprotein structured metalophosphatase with several defined functions. It is present in many tissues of all living beings from bacteria to mammals. The enzyme may catalyse the hydrolysis of various monophosphate esters at alkaline pH. The objective of this study was to quantify ALP functioning particularly in the membranes of eukaryotic cells. The membranes of seven different cells (myeloma cells; hybrid cells; erythroleukaemia cells; lymphocytes and erythrocytes) were tested for ALP activity using a cellular enzyme assay, which is based on the conversion of para-nitrophenylphosphate (p-NPP) to para-nitrophenol and the colorimetric determination of the resulting coloured product. The test system was optimised with respect to substrate concentration, reaction time and the number of cells used as a source of enzyme. The obtained values were converted to quantitative results through a standard curve created using commercial ALP. In order to determine the effect of serum concentration on enzyme activity, 1G2 hybridoma, which is among the cells used in this study and which synthesizes monoclonal antibody against human serum albumin, was produced in different serum concentrations ranging from 0 to 15%.

Cystic fibrosis transmembrane conductance regulator gene mutations in infertile males with congenital bilateral absence of the vas deferens.

Congenital bilateral absence of the vas deferens (CBAVD) is characterized by azoospermia and male infertility. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are associated with cystic fibrosis (CF), the most common autosomal recessive disorder in Caucasians. Recent publications on CBAVD raised the question whether CFTR gene mutations are responsible for CBAVD occurrence or not. This study was conducted to explore the role of CFTR gene mutations in the occurrence of CBAVD-dependent male infertility. Forty-four chromosomes of 22 CBAVD patients from Austrian ancestry were studied. For detection of the most common mutation DeltaF508, a deletion of phenylalanine at the 508th position of mature CFTR chloride channel protein, the 10th exon of the gene was screened by heteroduplex analysis. In order to identify non-DeltaF508 mutations, we also analyzed the entire coding regions, exon/intron boundaries of 27 exons and the 5'- and 3'-untranslated regions of the gene by denaturing gradient gel electrophoresis (DGGE) after polymerase chain reaction. All exons showing different banding patterns on the DGGE gels were sequenced to define existing DNA sequence variations. Among the analyzed 44 chromosomes of 22 patients, disease producing mutations were found in 31.8% (14/44). The most common mutation was DeltaF508 with a frequency of 43% (6/14), followed by R117H with 29% (4/14). Our results indicate that CFTR gene mutations are common but not the only reason for the occurrence of CBAVD-dependent male infertility. We recommend screening of the CFTR gene in these patients.