Glycosylation of procathepsin L does not account for species molecular-mass differences and is not required for proteolytic activity.

Cathepsin L is a major lysosomal cysteine proteinase in mouse and human cells. Despite similar predicted molecular masses, procathepsin L in these two species migrates on SDS/polyacrylamide gels with apparent molecular masses of 39 kDa and 42 kDa respectively. To determine if glycosylation differences account for this discrepancy, and to ascertain whether glycosylation is essential for enzymic activity, mouse and human procathepsins L were expressed at high concentrations in mouse NIH 3T3 cells or in human A431 cells after DNA-mediated transfection of cloned DNAs for these enzymes. In pulse-chase studies, human procathepsin L transfectants synthesized and secreted large amounts of enzymically active 42 kDa proenzyme and processed it into 34 kDa and 26 kDa intracellular peptides, a pattern of secretion and processing similar to that seen with endogenous or transfected mouse procathepsin L. Both translation of cloned procathepsin L cDNAs in vitro and Endoglycosidase H treatment of 39 kDa mouse and 42 kDa human procathepsin L resulted in non-glycosylated proteins 2 kDa lower in molecular mass than the untreated proteins for both species. This suggests that glycosylation differences are not responsible for the molecular-mass disparity between the two species. Moreover, Endoglycosidase H-treated mouse enzyme retained full proteolytic activity, indicating that glycosylation of cathepsin L is not essential for enzymic function.     

Neutron fibre diffraction study of DNA hydration

Interactions with water are crucial to the conformation assumed by the DNA double helix. The location of water around the D conformation has been investigated in a neutron fibre diffraction study which shows that water is ordered in the minor groove of the DNA. The D conformation is important since its occurrence is limited to specific DNA base pair sequences which have been identified as functionally significant. This study is of particular interest because the D conformation has not been reported in single crystal studies of oligonucleotides.     

Effects of plasmid presence on growth and enzyme activity of Escherichia coli DH5α

Summary The effects of recombinant DNA propagation and gene expression on the physiology of the host cell was investigated using a series of copy number mutant plasmids. The plasmids at copy numbers of 30, 57, 115 and 501 per chromosome equivalent encoded constitutive production of the enzyme -lactamase. Ribose phosphate isomerase activity was relatively unaffected by plasmid presence, and glucose-6-phosphate dehydrogenase, fructose 1,6-diphosphate aldolase and fructose 1,6-diphosphatase activities were lower in plasmid-containing cells than in the plasmid-free host strain. Increasing copy number resulted in increased depression of enzyme activity levels. The results indicate that plasmid presence mediates subtle changes in the net expression of host enzymes involved in carbon metabolism. Responses of Escherichia coli DH5 in Evans medium to these plasmids differed substantially from responses of E. coli HB101 in rich medium.Offprint requests to: J. E. Bailey     

The cDNA cloning of a pea (Pisum sativum) seed lipoxygenase. Sequence comparisons of the two major pea seed lipoxygenase isoforms.

Cloning and sequencing of two cDNAs from mRNA of maturing pea (Pisum sativum) seeds allowed the deduction of the complete amino acid sequence of a lipoxygenase polypeptide which is most similar to that of soya-bean lipoxygenase 2. The predicted Mr of this polypeptide is 97134, and its sequence permits comparisons between the lox2-type and the lox3-type lipoxygenase isoforms from pea and soya bean (Glycine max).     

Nucleotide and deduced amino acid sequence of the hydrophobic surfactant protein SP-C from rat: expression in alveolar type II cells and homology with SP-C from other species

Pulmonary surfactant lowers surface tension in the lung. Its deficiency leads to the severe physiologic abnormalities seen in the respiratory distress syndrome. The hydrophobic surfactant proteins, SP-B and SP-C, appear to be especially important in the surface-spreading characteristics of pulmonary surfactant. We report the nucleotide sequence of cDNA clones for rat SP-C and compare the deduced amino acid sequence for SP-C from several species. A highly conserved domain exists within the confines of mature human SP-C. An Eisenberg plot of this region predicts a membrane-associated helix. We also demonstrate by Northern analysis the tissue-specific expression of SP-C. A comparison of signal strength between total lung RNA and RNA derived from isolated type II cells supports the idea that most SP-C messenger RNA in total lung can be accounted for by that present in alveolar type II cells.     

Diffusional dynamics of an active rhodamine-labeled 1,4-dihydropyridine in sarcolemmal lipid multibilayers.

A "membrane bilayer pathway" model, involving ligand partition into the bilayer, lateral diffusion, and receptor binding has been invoked to describe the 1,4-dihydropyridine (DHP) calcium channel antagonist receptor binding mechanism. In an earlier study (Chester et al. 1987. Biophys. J. 52:1021-1030), the diffusional component of this model was examined using an active fluorescence labeled DHP calcium channel antagonist, nisoldipine-lissamine rhodamine B (Ns-R), in purified cardiac sarcolemmal (CSL) lipid multibilayers. Diffusion coefficient measurements on membrane-bound drug and phospholipid at maximum bilayer hydration yielded similar values (3.8 x 10(-8) cm2/s). However, decreases in bilayer hydration resulted in dramatically reduced diffusion coefficient values for both probes with substantially greater impact on Ns-R diffusion. These data suggested that hydration dependent diffusional differences could be a function of relative probe location along the bilayer normal. In this communication, we have addressed the relative effect of the rhodamine substituent on Ns-R diffusion complex by examining the diffusional dynamics of free rhodamine B under the same conditions used to evaluate Ns-R complex and phospholipid diffusion. X-ray diffraction studies were performed to determine the Ns-R location in the membrane and model the CSL lipid bilayer profile structure to give a rationale for the differences in probe diffusional dynamics as a function of interbilayer water space.     

The oxidation of the calcium probe quin2 and its analogs by prostaglandin H synthase

Quin2 and its analogs BAPTA, 5,5'-dimethyl BAPTA, 5,5'-difluoro BAPTA, fura-2, and indo-1 were developed to measure intracellular calcium concentrations. In this study we investigated whether quin2 and its analogs are susceptible to peroxidase-catalyzed oxidation. The hydroperoxidase activity of prostaglandin H synthase, like other peroxidases, is capable of oxidizing a wide variety of substrates. It was found that quin2 and its analogs served as reducing cofactors for the hydroperoxidase activity of prostaglandin H synthase, undergoing oxidation in the process. Furthermore, arachidonic acid metabolism was stimulated. Oxidation of quin2 and its analogs resulted in the formation of a carbon-centered radical, as could be detected by ESR, and in the formation of formaldehyde. Quin2 fluorescence decreased upon addition of arachidonic acid and prostaglandin H synthase. Furthermore, addition of calcium no longer resulted in an increase in quin2 fluorescence, as was observed prior to the addition of arachidonic acid and the enzyme. This indicates that one or more of the -N-CH2-COOH groups, which are responsible for the binding of calcium, were oxidized by the hydroperoxidase. Since prostaglandin H synthase is present in many cellular systems in which calcium concentrations are modulated, oxidation of the calcium probe might not only affect the measurement of intracellular calcium but could activate arachidonic acid metabolism as well.     

Distribution of glucose carbons to metabolic products in the perfused rat hindlimb over a range of glucose concentrations

To determine the distribution of glucose carbons to the major metabolic pathways as a function of glucose and insulin concentrations in fed rat muscle, rat hindlimbs were perfused with and without 100 nM insulin in the presence of 1-20 mM [U-14C]-D-glucose. The incorporation of radioactivity into CO2, anionic metabolites released into the medium and muscle cell fractions were determined. The results indicate that the major metabolic fate of glucose taken up by muscle in the fed rat is conversion to anions released into the medium and that this process is saturable with increasing glucose concentrations. The data suggest that saturation of the glycolytic pathway is the major cause of the previously observed saturation of the glucose disposal rate associated with increasing glucose concentration.     

Isolation and chromosomal localization of the human glutathione peroxidase gene

We have isolated cDNA clones for the gene, termed GPX1, encoding the major human selenoprotein, glutathione peroxidase. Sequence analysis confirmed previous findings that the unusual amino acid seleno-cysteine is encoded by the opal terminator codon UGA. Southern blot analysis of human genomic DNA with the GPX1 cDNA showed that restriction endonucleases without sites in the probe sequence produced three hybridizing bands at standard stringency, diminishing to one strongly and one weakly hybridizing band at high stringency. In situ hybridization localized the human GPX1 gene to a single site on chromosome 3, at region 3q11-13.1. Thus, three genomic sites bear sequence homology to the GPX1 cDNA, and the one most homologous maps to 3q11-13.1.