Secondary structures of rat lipolytic enzymes: circular dichroism studies and relation to hydrophobic moments

To explore the secondary structures of lingual and pancreatic lipases, circular dichroism measurements were performed. Maximum average ellipticities were used to calculate the percentage of alpha-helices, beta-sheets, and random coils. Lingual lipase had an ellipticity of -20235 +/- 140 deg cm2/dmol (mean +/- SE) at 220 nm suggesting 60% alpha-helix, 20% beta-sheet and 20% random coil structure, but the mean ellipticity for pancreatic lipase was -14093 +/- 82 deg cm2/dmol (mean +/- SE) at 210 nm suggesting a 34.8% alpha-helical, 25% beta-sheet and 40% random coil secondary structure. An alpha-helical stretch of residues with a large hydrophobic moment ("globular" alpha-helix by hydrophobic moment plot) from amino acids 382 through 389 at the COOH-terminal end of lingual lipase was noted. This sequence, absent in pancreatic lipase, may account for the avid binding of lingual lipase to fat emulsion particles.     

Some mathematical aspects of mapping DNA cosmids

A number of experimental and mathematical problems must be solved before high resolution physical maps of mammalian chromosomes can be reliably determined. Such a map might consist of an ordered set of nonsequenced, overlapping DNA fragments 20,000-40,000 bases long, produced by digestion of a chromosome, using two restriction enzymes. Map construction requires assigning a signature to each fragment that differentiates it unambiguously from every other fragment, and then devising a computationally efficient algorithm that will provide a unique ordering of the fragments. In the first part of this paper we present a polynomial time algorithm that yields a unique map, and is largely independent of the method for assigning signatures. In the next section we analyze the distribution of lengths of restriction digest fragments and discuss the implications for the algorithm, including the expected number of map gaps. Finally, we discuss a specific method for assigning signatures proposed by Hans Lehrach, based on which of a panel of probes binds to a given fragment. In particular we examine the effects of fragment length heterogeneity on the theoretical optimum length and number of probes, and the extent to which false signatures might be obtained by nonspecific binding. We conclude that the Lehrach strategy is effective provided the number of probes is >-150, but that each fragment will need testing with at most 25 probes.     

The amyloid percursor protein of Alzheimer disease is expressed as a 130 kDa polypeptide in various cultured cell types

The vascular and parenchymal amyloid deposits in Alzheimer disease (AD), normal aging and Down syndrome are mainly composed of a 4 kDa polypeptide (A4), which derives from a larger precursor protein (APP). There is evidence that APP is a transmembrane glycoprotein present in most tissues, but the characteristics of APP in intact cells are not yet known. In order to investigate this issue, we examined the immunoreactivity of fibroblasts of human and nonhuman cell lines with antisera raised to synthetic peptides corresponding to A4 and to two other domains of the APP. All three antisera recognized a 130 kDa polypeptide (APP-130) in immunoblots from all cell lines. In fibroblasts, an additional polypeptide of 228 kDa (APP-228) was recognized by the antiserum to A4. In immunoblots of two dimensional gels, APP-130 showed a pI of 6.2, while APP-228 failed to focus in the pH range of 4.7-7.0. Sequential extractions of cells with buffer and with Triton X-100 indicate that APP-130 is extractable with nonionic detergents at high ionic strength, whereas 228 kDa APP is a cystolic component. Immunofluorescence staining is consistent with an intracellular perinuclear and plasma membrane localization. It is concluded that APP-130 and APP-228 are two forms of the APP which result from extensive posttranslational modifications of a smaller original gene product. It is likely that APP undergoes similar posttranslational modifications in different cell types.     

The cationic composition of incubated cerebral cortex slices

1. A new type of cutting table is described. It makes use of the elastic properties of a nylon thread, 0.08 mm thick, in which longitudinal vibrations greatly increase its ability to cut through soft tissue. Two slices of cerebral cortex may thus be obtained within 3-4 min after the death of an animal. 2. The extent of the swelling of a brain slice, as well as the ionic shift, is directly related to the amount of oxygen in the incubating fluid. Under the best conditions of oxygen supply, the swelling of a first slice was close to 12.2 +/- 4.3 per cent after 5 hr of incubation. The corresponding values for the Na+ and K+ contents were respectively 119.3 +/- 6.9 and 70.9 +/- 5.0 microequiv./g of final fresh weight (P2). 3. Incubation in complete anoxia leads to a considerable shift in the cation content of the slice, which acquires a composition close to that of the incubating fluid. This suggests that a large part of the cell population is still metabolically active when oxygen is present. 4. The inulin space represents 47.1 +/- 5.8 per cent of the initial fresh weight. It is independent of the amount of fluid taken up by the slice as well as of anoxia. 5. The cation content of the non-inulin space, calculated by assuming that the inulin space has the same composition as the incubation medium, was 77.7 microequiv./ml and 160.3 microequiv./ml for Na+ and K+ respectively. 6. The meaning of the inulin space, as well as the physico-chemical state of the cations in the slice, are discussed.     

Monte Carlo study of the effect of β2-microglobulin on the binding cleft of the HLA-A2 complex

Peptide recognition by class I products of the major histocompatibility complex requires association of the class I heavy chain with β₂-microglobulin. We present results of Monte Carlo simulations of the β-pleated sheet floor of the human class I MHC molecule, HLA-A2, with and without β₂-microglobulin. We find a significant effect of β₂-microglobulin on the side chains of residues near a region that would accommodate the C-terminus of a bound peptide. By modeling simultaneously each loop and its neighboring strand at either end of the class I cleft, we find that β₂-microglobulin restricts the conformational space of residues that are central to binding peptides. The effect is most pronounced for R97 and H114 and somewhat less important for Y99 and Y116, the latter forming strong hydrogen bonds with neighboring residues in the heavy chain itself.     

Evolution of the ATP-binding-cassette transmembrane transporters of vertebrates

The ATP-binding-cassette transmembrane transporters (ABC transporters) known from vertebrates belong to four major subfamilies: (1) the P- glycoproteins (Pgp); (2) the cystic fibrosis transmembrane conductance regulators (CFTR); (3) the Tap proteins encoded with the major histocompatibility complex of mammals; and (4) the peroxisomal membrane proteins. Both Pgp and CFTR have a structure suggesting a past internal gene duplication; a phylogenetic analysis indicated that these duplications occurred independently, while an independent tandem gene duplication occurred in the case of the Tap family. Both the Pgp and Tap proteins show evidence of relationship to bacterial ABC transporters lacking internal duplication, and both are significantly more closely related to the HlyB and MsbA families of transporters from purple bacteria than they are to ABC transporters from nonpurple bacteria. The simplest hypothesis to explain this observation is that eukaryotic Pgp and Tap genes are descended from a mitochondrial gene or genes that were subsequently translocated to the nuclear genome. The Pgp genes of eukaryotes are characterized by a remarkable degree of convergent evolution between the ATP-binding cassettes of their N- terminal and C-terminal halves, whereas no such convergence is seen between the two halves of CFTR genes or between the duplicated Tap genes. Exon 13 of the CFTR gene, which encodes a putative regulatory domain not found in other ABC transporters apart from CFTR, showed high levels of both synonymous and nonsynonymous difference in comparisons among different mammalian species, suggesting that this region is a mutational hot spot.      

Evidence for the presence of β-lactamase inStreptomyces glaucescens and its inhibition by sodium clavulanate

Streptomyces glaucescens is shown to possess -lactamase activity which is inhibitable by clavulanate. This is important in regard to its use as a cloning host for enzymes of \-lactam biosynthesis.