Carboxyl-terminal sequence of entactin deduced from a cDNA clone.

Entactin is a widely distributed basement membrane sulfated glycoprotein of approximately equal to 150 kDa. The entactin gene is expressed early in mouse embryogenesis. Two cDNA clones complementary to rat entactin mRNA were isolated by antibody screening of an oligo(dT)-primed cDNA library constructed in the lambda gt11 expression vector. One of the clones, lambda 1E, was subcloned into plasmid pBR322 and further characterized. The clone contained sequences complementary to an mRNA species 6 kilobases in length. This mRNA was translated in rabbit reticulocyte lysates to yield a polypeptide of 143 kDa that was precipitated with anti-entactin antiserum. The cDNA insert, 1328 base pairs long, was sequenced and found to contain an open reading frame of 729 base pairs that coded for 243 amino acids at the carboxyl terminus of entactin. Analysis of the peptide revealed no extended alpha-helical or beta-sheet secondary structures. Radiolabeled probes prepared by nicktranslation of p lambda 1E were used to monitor the steady-state levels of entactin mRNA in F9 embryonal carcinoma cells that were induced to differentiate by exposure to retinoic acid and dibutyryl cyclic AMP. The increase in steady-state levels of entactin mRNA lagged behind the increase in mRNA for the B2 chain of laminin, suggesting that laminin and entactin are independently rather than coordinately regulated.

In vitro formation of fine fibrils with a D-periodic banding pattern from type V collagen.

Type V collagen was isolated from human placenta by limited pepsin treatment and purified by salt fractionation. A solution of type V collagen was dialyzed at 4 degrees C against phosphate-buffered saline or against 0.02 M Na2HPO4. Aggregates formed under these in vitro conditions from a pure type V collagen solution were examined by electron microscopy. The aggregates were fine flexible fibrils. The fibrils formed during incubation at 25 degrees C were of relatively uniform diameter, 34.8 +/- 9.1 nm and did not show an axial banding pattern. When the specimen was incubated at 37 degrees C, the fibrils were of slightly larger diameter, 38.2 +/- 9.1 nm and almost all the fibrils had the axial repeat pattern of 67 nm. The ability of type V collagen to form banding fibrils is discussed in relation in the localization of the collagen in tissues.

Variations in chemical composition of human glomerular and tubular basement membranes with age and disease.

A graded sieving procedure was used to isolate glomeruli and tubules from renal cortex of men of premature age up to 80 years and of 4 patients suffering from Zellweger syndrome, congenital nephrotic syndrome, polycystic renal disease or diabetes mellitus. Glomerular and tubular basement membranes (GBM and TBM, respectively) were obtained with a detergent procedure. Purity of basement membrane preparations was controlled with light and electron microscopy and by estimating total phosphorus content. Amino acid and carbohydrate composition of the basement membranes were determined and statistically evaluated. Comparison of GBM and TBM from the same kidneys showed at all ages that GBM contains more 3-hydroxyproline, neuraminic acids and mannose. These differences may contribute to the different immunogenic properties of the two basement membranes reported in the literature. Significant changes with age in the chemical composition were found, suggesting that the proportion of collagenous peptide moieties increases and that of noncollagenous peptide moieties decreases with age in both GBM and TBM. In addition, the hydroxylation grade of proline and lysine increases significantly with age reaching an adult level for GBM after 4-6 months of age and for TBM at late childhood. The age-related changes in basement membrane composition may influence functional properties of these extracellular renal structures. The chemical composition of GBM and/or TBM of the 4 patients showed some differences in comparison to control preparations from persons with ages approximating that of the patients.

Phospholipid of the rat glomerular basement membrane in experimental nephrosis.

Phospholipids were found to be a constant component of rat glomerular basement-membrane preparations. The concentration fell during preparation of basement membrane by sonication of whole glomeruli, but then remained constant despite continued sonication. The proportions of the individual phospholipids were different from those of whole renal tissue or of isolated glomeruli. The basement-membrane preparations had no (Na(+)+K(+))-activated adenosine triphosphatase activity, an enzyme that is bound to plasma membranes. The concentration of lipid P was decreased on exposure in vivo or in vitro to antiserum against basement membrane; 7 days after injection of antiserum there was a change in the phospholipid composition, with a relative increase in phosphatidylcholine and a decrease in sphingomyelin content. The metabolic turnover rate of the lipid P remaining in the membrane was normal, as determined by (32)P incorporation. The loss of phospholipid was associated with decreases in the relative concentrations of hydroxyproline, hydroxylysine and glycine, and relative increases in proline, lysine, serine, threonine and valine. Administration of aminonucleoside and daunomycin produced proteinuria but did not cause a decrease in lipid P. Anticollagen and anti-lymphocyte sera that attached to the basement membrane but failed to produce proteinuria, also failed to affect the phospholipid content.