Homology between antigenic determinants of bovine clathrin and clathrin from the brown alga Laminaria digitata

Coated vesicles, essential organelles of intracellular membrane traffic, have been extensively studied in animal and higher plant cells. In the algae, cytological studies only have been performed which demonstrate the presence of such coated vesicles with their surrounding clathrin lattice. The present work has been carried out on coated vesicles isolated for the first time from the brown algae Laminaria digitata. For comparison of the antigenic characteristics of clathrin prepared from the Bovine brain or adrenocortical cells and the clathrin prepared from algae, polyclonal antibodies have been raised to a purified Bovine brain clathrin in Goat and to Bovine adrenocortical clathrin in Rabbit. The positive immunological responses of the coated vesicles and the clathrin from Algae to these antibodies, evidence an homology between antigenic determinants of clathrin from animal and vegetal cells.     

Size and composition of lymph chylomicrons following feeding corn oil or its fatty acid methyl esters

Male rats with thoracic duct cannulae were intubated with corn oil or fatty acid methyl esters and the lymph was collected over the next 2-72 h. The apoprotein (apo) composition of the chylomicrons, isolated by conventional ultracentrifugation, was determined by sodium dodecyl sulfate - polyacrylamide - glycerol gel electrophoresis and isoelectric focusing. The lipid content and composition was assessed by gas--liquid chromatography. The particle size was obtained by calculation and confirmed by electron microscopy. The study demonstrates that both the monoacylglycerol (corn oil feeding) and the phosphatidic acid (methyl ester feeding) pathways of triacylglycerol biosynthesis yield chylomicrons with closely similar apoprotein profiles representing apo B-48, apo A-IV, apo E, apo A-I, and the apo C components. A protein band corresponding to apo B-100 was occasionally observed as a minor component of the chylomicrons from both groups of animals. The chylomicrons from corn oil feeding had about two times larger diameters than those from methyl ester feeding. There were no significant differences in the composition of the apoproteins, although the smaller particles had two times higher apoprotein/triacylglycerol ratios. It was calculated that the amount of apo B per lipid particle for the ester fed rats ranged from one to eight molecules and was closely correlated with the particle size. The corn oil fed rats yielded about three molecules apo B per lipid particle regardless of the particle size. It is concluded that the pathway of intestinal triacylglycerol biosynthesis has a significant effect on the apoprotein mass and to a lesser extent on the apoprotein and lipid composition of the chylomicrons. The phosphatidic acid pathway produces smaller particles and transfers to the bloodstream twice as much apoprotein per gram of fat than the monoacylglycerol pathway, which yields the larger particles. Possible variations in the site and rate of biosynthesis of the triacylglycerols could not be entirely excluded as contributing factors.     

Stereochemical course of intestinal absorption and transport of mustard-seed oil triacylglycerols in the rat

Male rats with thoracic duct cannulae were intubated with mustard-seed oil or the corresponding fatty acid methyl esters and the lymph was collected over 0-24 h. The chylomicron and very low density lipoprotein fractions were obtained by conventional ultracentrifugation. The triacylglycerols and glycerophospholipids were isolated and the positional distribution and molecular association of fatty acids were determined by stereospecific and chromatographic methods. The oleic, linoleic, and linolenic acids were recovered in the lymph in the proportion in which they occurred in the fat fed, while eicosenoic, erucic, and lignoceric acids were rejected to about the same extent by the two pathways of intestinal triacylglycerol biosynthesis. It is shown that the lymph triacylglycerols arising via the monoacylglycerol or the phosphatidic acid pathway possess structures that are closely similar to each other and to that of the original mustard-seed oil. It is proposed that this is a result of comparable fatty acid and positional specificity of the acyltransferases associated with the acylglycerol synthesis in the animal and plant tissues and the wide range of fatty acid chain lengths in the mustard-seed oil.     

Phospholipid methylation in canine myocardium: kinetic characteristics and the effect of endotoxin administration

The kinetic characteristics and the effect of endotoxin administration on the enzymatic methylation of phospholipids in dog heart microsomes were studied using S-adenosyl-L-[methyl-3H]methionine as a methyl donor. Kinetic studies in control dogs reveal that the stepwise methylation of phosphatidylethanolamine to phosphatidylcholine was catalyzed by three different enzymes. Methyltransferase I catalyzed the methylation of phosphatidylethanolamine to phosphatidyl-N-monomethylethanolamine, had a very low Km (approximately 1.5 microM) for S-adenosylmethionine, and a pH optimum of 6.5, and it was stimulated by Mg2+ and Ca2+. Methyltransferase II catalyzed the methylation of phosphatidyl-N-monomethylethanolamine to phosphatidyl-N,N-dimethylethanolamine, had a low Km (8-12 microM) for S-adenosylmethionine, and a pH optimum of 8.5, and it was stimulated by low concentrations (less than 1 mM) of Ca2+ but was unaffected by Mg2+. Methyltransferase III catalyzed the formation of phosphatidylcholine from phosphatidyl-N,N-dimethylethanolamine, had a high Km (approximately 33 microM) for S-adenosylmethionine, and a pH optimum of 9.5, and it was unaffected by Mg2+ or Ca2+. Experiments with trypsin digestion indicate that methyltransferases I and III were partially embedded while methyltransferase II was completely exposed to the surface of the membrane. Endotoxin administration (2 and 4 hr) decreased the Km and Vmax by 30 to 36% and 24 to 37.7%, respectively, for S-adenosylmethionine. Since the enzymatic methylation of phospholipids has been implicated to play an important role in the regulation of membrane structure and function, the endotoxin-induced decreases in the Km and Vmax of phospholipid-methylating enzymes in dog heart microsomes may contribute to the development of myocardial dysfunction in endotoxin shock.     

Characterization of complementary deoxyribonucleic acid for human adrenocortical 17 alpha-hydroxylase: a probe for analysis of 17 alpha-hydroxylase deficiency

To provide a basis for investigation of the molecular mechanisms underlying the hormonal regulation of steroid 17 alpha-hydroxylase (P-450 17 alpha) activity in adrenal, ovary, and testis as well as human 17 alpha-hydroxylase deficiency, we have isolated from a human fetal adrenal cDNA library a cDNA sequence complementary to the mRNA that encodes the human P-450 17 alpha enzyme. Of 75,000 colonies from the library that were screened by use of a nick-translated 5'-specific bovine P-450 17 alpha cDNA probe, 10 positive colonies were isolated and the clone with the longest insert (pcD-17 alpha H) was selected for further characterization. pcD-17 alpha H encodes the complete human P-450 17 alpha protein having approximately 78% homology at the nucleotide level and 71% homology at the amino acid level when the sequence of pcD-17 alpha H is compared to the bovine P-450 17 alpha cDNA sequence. By transient expression of the human P-450 17 alpha cDNA clone in COS 1 cells, we have demonstrated that the 17 alpha-hydroxylase and 17,20 lyase activities reside within the same human P-450 17 alpha polypeptide chain. The insert was also used as a probe to investigate, by means of Southern blot analysis, possible alterations in the P-450 17 alpha gene sequence in DNA isolated from skin fibroblasts from three patients with clinically characterized 17 alpha-hydroxylase deficiencies. No changes were detected in the DNA of any of the patients by this analysis.     

Patterns of polymorphism and linkage disequilibrium for cystic fibrosis

Four polymorphic markers that map within 80 kb of an HTF island which is genetically very close to the cystic fibrosis locus have been identified. We have analyzed the linkage disequilibrium between each of these markers and the cystic fibrosis mutation in 89 families from four European countries, Denmark, Finland, Spain, and Great Britain. Strong linkage disequilibrium between three polymorphic sites and cystic fibrosis was observed. The markers on the J3.11 (D7S8) side of the HTF island show stronger disequilibrium than those on the met side. Linkage disequilibrium between markers and disease alters the probability that a person of a given haplotype is a carrier in some populations and helps to identify regions of a sequence that are most likely to contain the cystic fibrosis mutation.     

Plasmid-encoded copper resistance and precipitation by Mycobacterium scrofulaceum.

A copper-tolerant Mycobacterium scrofulaceum strain was able to remove copper from culture medium by sulfate-dependent precipitation as copper sulfide. Such precipitation of copper sulfide was not observed in a derivative that lacks a 173-kilobase plasmid. In addition, the plasmid-carrying strain has a sulfate-independent copper resistance mechanism.     

Conformational predictive studies on the activation segment of pancreatic procarboxypeptidases

Little is known about the conformation and evolutionary origin of the activation segment of pancreatic procarboxypeptidases. Analysis of the sequence and secondary structure propensities of these propeptide segments indicate that they contain two regions structurally related to the Ca2+-binding sites of the EF-hand protein family. This proposed homology could explain how (and why) carboxypeptidases developed such long (94 residues) activation peptides.     

Microheterogeneity of the malate dehydrogenase from several sources

Different homogeneously purified cytosolic malate dehydrogenases gave, on isoelectric focusing, several active bands. The phenomenon could not be assigned to differences in their molecular weights or to alterations in the enzyme preparations during the purification procedure. Resolution of the multiple malate dehydrogenase active bands was achieved by chromatofocusing. The aged isolated subforms always yielded the original electrofocusing pattern. This fact suggests that conformational isomerism is a likely explanation for the charge heterogeneity of the enzymes studied.     

Enzymatic transformation of PGH2 to PGF2 alpha catalyzed by glutathione S-transferases

Glutathione S-transferases (GSTs) purified from both rat liver cytosol and microsomes catalyzed the direct reduction of PGH2 to PGF2 alpha. As much as 40% of the substrate was transformed into a prostanoid whose Rf value corresponded to that of PGF2 alpha. The identification of the reaction product as PGF2 alpha was confirmed by TLC and reverse-phase HPLC as well as by mass spectral analysis. In the absence of GSTs, PGH2 was found to be primarily converted to PGE2 and PGD2. Also, PGF2 alpha formation was completely abolished by decylglutathione, a potent inhibitor of both peroxidase and transferase activity associated with GSTs. These results indicate that the direct reduction of endoperoxide moiety of PGH2 to form PGF2 alpha is an enzymatic process. Interestingly, selenium-dependent glutathione peroxidase (Se-GSH-Px) showed very little PGF2 alpha formation from PGH2. However, this enzyme was very active in the reduction of PGG2 to PGH2. In contrast, GSTs were very poor in the conversion of PGG2 to PGH2. Therefore, it is possible that the relative tissue distribution of Se-GSH-Px and GSTs might play an important role in the tissue specific synthesis of PGF2 alpha.