Peptidases II. Localization of dipeptidylpeptidase IV (DPP IV). Histochemical and biochemical study]

Fresh frozen, unfixed, chloroforme-acetone treated or freeze-dried cryostat sections or sections from aldehyde-fixed blocks of tissue were tried for the histochemical investigation of dipeptidylpeptidase IV (DPP IV) with L-glycyl-L-prolyl(gly-pro)-naphthylamides as substrates and stable or unstable diazonium salts for simultaneous coupling and various buffers, pH 5--7.5 in rats, mice, guinea-pigs, cats, rabbits, hamsters and human enterobiopsies. The best results are obtained with 1.7--3.4 mM gly-pro-4-methoxy-2-naphthylamide and 1 mg Fast Blue B/ml or (with some limitations) 0.025 ml hexazotized new fuchsine/ml in 0.1 M cacodylate or phosphate buffer, pH 7.5 and unfixed sections for the demonstration of the total activity of DPP IV and freeze-dried celloidin-mounted cryostat sections for the precise localization of the enzyme or the detection of lysosomes, Golgi apparatus and secretion granules sections from aldehyde fixed tissue blocks are only suitable to study the lysosomal hydrolysis of gly-pro-naphthylamides between pH 5 and 7 when hexazotized p-rosaniline or new fuchsine are employed. DPP IV is firmly bound to strutures and shows species- and organ-dependent differences. In general, the enzyme occurs in the capillary endothelium, sinusoidal cells, perineurium, epithelial cells of intercalated and striated ducts, microvillous zone of intestinal crypts and villi, uterus, Fallopian tubes, ductus epididymis and proximal renal tubules, hepatocyte and lymphocyte membrane, plasmalemma of pseudostratified and transient epithelia and in the capsules and interstitium of many organs. These sites of activity can be completely inhibited by diisopropyl fluorophosphate and partially by Pb2+; Mg2+, Mn2+, Co2+ EDTA are without any influence. Phenantrolin may activate DPP IV. The biochemical assay works with 10 mM gly-pro-2-naphthylamide in 0.1 M cacodylate buffer, pH 7; the enzyme activity is determined fluorometrically in guinea-pig and rat organs; the data confirm and enlarge the species- and organ-dependent differences revealed by histochemistry. Compared with other dipeptide as well as tripeptide and amino acid naphthylamides the results obtained for DPP IV suggest a peptidylpeptidase which seems to be involved in other metabolic processes beside the degradation of collagen.     

Genetic studies of human apolipoproteins. IV. Structural heterogeneity of apolipoprotein H (beta 2-glycoprotein I).

Human beta 2-glycoprotein I has recently been identified as a component of several human plasma lipoprotein fractions and therefore termed as apolipoprotein H. Its metabolic function in lipid metabolism is not known with certainty, though it may be involved in very-low-density-lipoprotein metabolism. Previously, inherited quantitative variation in beta 2-glycoprotein I has been suggested in man. In this investigation, we document the evidence of genetically determined structural polymorphism of apolipoprotein H or beta 2-glycoprotein I by using thin-layer polyacrylamide isoelectric focusing gels followed by immunological identification by double antibody staining. The apolipoprotein H structural locus is characterized by the occurrence of three common alleles in U.S. whites and blacks. The frequency distributions of the three alleles designated APO H1, APO H2, and APO H3 are .059, .882, and .059 in whites and .017, .902, and .068 in blacks, respectively. In addition, the gene product of a fourth allele, APO H4, has been observed at polymorphic frequency in black individuals and may represent a black marker variant. Family data confirm the hypothesis of four alleles at a single APO H gene locus with an autosomal codominant pattern of inheritance.     

Genetic and biochemical characterization of the Lactobacillus delbrueckii subsp. lactis bacteriophage LL-H lysin.

LL-H, a virulent phage of Lactobacillus delbrueckii subsp. lactis, produces a peptidoglycan-degrading enzyme, Mur, that is effective on L. delbrueckii, Lactobacillus acidophilus, Lactobacillus helveticus, and Pediococcus damnosus cell walls. In this study, the LL-H gene mur was cloned into Escherichia coli, its nucleotide sequence was determined, and the enzyme produced in E. coli was purified and biochemically characterized. Mur was purified 112-fold by means of ammonium sulfate precipitation and cation-exchange chromatography. The cell wall-hydrolyzing activity was found to be associated with a 34-kDa protein. The C-terminal domain of Mur is not essential for catalytic activity since it can be removed without destroying the lytic activity. The N-terminal sequence of the purified lysin was identical to that deduced from the nucleotide sequence, but the first methionine is absent from the mature protein. The N-terminal part of this 297-amino-acid protein had homology with several Chalaropsis-type lysozymes. Reduction of purified and Mur-digested L. delbrueckii cell wall material with labeled NaB3H4 indicated that the enzyme is a muramidase. The temperature optimum of purified Mur is between 30 and 40 degrees C, and the pH optimum is around 5.0. The LL-H lysin Mur is stable at temperatures below 60 degrees C.     

Detection of genetic variation with radioactive ligands. IV. X-linked, polymorphic genetic variation of thyroxin-binding globulin (TBG).

A genetically determined, polymorphic electrophoretic variant of thyroxin-binding alpha-globulin (TBG) is found in sera from populations of African and Oceania origin, although not in Caucasians nor Orientals. The TBG polymorphism is inherited in X-linked fashion, based on data from American blacks, and thus provides an X-chromosome marker with a relatively high gene frequency in this ethnic group (frequency of the slow allele, TBGs, is 11%). This slow variant should prove valuable in expanding the map of the X chromosome and in linkage studies. An additional family exhibiting X-linked TBG deficiency is also described.     

Prenatal diagnosis of hypophosphatasia; genetic, biochemical, and clinical studies.

This report has considered three approaches to the prenatal diagnosis of the severe, early onset form of hypophosphatasia. Two of these approaches, ultrasonography and the determination of the bone/liver isozymes of alkaline phosphatase (ALP) in cultured amniotic fluid cells, have proven useful diagnostically. The third method, assay of the bone/liver isozyme activity or total activity in supernatant amniotic fluid, was not informative for the affected fetus we studies. Failure to visualize a well-defined fetal skull after 16 weeks of pregnancy when the level of alpha-fetoprotein in the amniotic fluid is normal should arouse the suspicion of hypophosphatasia. Because the disease is known to manifest clinical variabiltiy, studies to detect both the biochemical defect as well as the structural manifestations should be considered. The combined use of ultrasonography, analysis of amniotic fluid alpha-fetoprotein, and the measurement of the bone/liver ALP in cultured amniotic fluid cells would appear to be the best approach to the prenatal diagnosis.     

Structural analysis of the GLUT1 facilitative glucose transporter (review).

The structure of the human erythrocyte facilitative glucose transporter (GLUT1) has been intensively investigated using a wide array of chemical and biophysical approaches. Despite the lack of a crystal structure for any of the facilitative monosaccharide transport proteins, detailed information regarding primary and secondary structure, membrane topology, transport kinetics, and functionally important residues has allowed the construction of a sophisticated working model for GLUT1 tertiary structure. The existing data support the formation of a central aqueous channel formed by the juxtaposition of several amphipathic transmembrane-spanning alpha-helices. The results of extensive mutational analysis of GLUT1 have elucidated many of the structural determinants of the glucose permeation pathway. Continued application of currently available technologies will allow further refinement of this working model. In addition to providing insights into the molecular basis of both normal and disordered glucose homeostasis, this detailed understanding of structure/function relationships within GLUT1 can provide a basis for understanding transport carried out by other members of the major facilitator superfamily.     

Ethylene thiourea (ETU). A review of the genetic toxicity studies

Ethylene thiourea (ETU) is a common contaminant, metabolite and degradation product of the fungicide class of ethylene bisdithiocarbamates (EBDCs); as such, they present possible exposure and toxicological concerns to exposed individuals. ETU has been assayed in many different tests to assess genotoxicity activity. While a great number of negative results are found in the data base, there is evidence that demonstrates ETU is capable of inducing genotoxic endpoints. These include responses for gene mutations (e.g. Salmonella), structural chromosomal alterations (e.g. aberrations in cultured mammalian cells as well as a dominant lethal assay) and other genotoxic effects (e.g. bacterial rec assay and several yeast assays).It is important to consider the magnitude of the positive responses as well as the concentrations/doses used when assessing the genotoxicity of ETU. While ETU induces a variety of genotoxic endpoints, it does not appear to be a potent genotoxic agent. For example, it is a weak bacterial mutagen in the Salmonella assay without activation in strain TA1535 at concentrations generally above 1000 μg/plate. Weak genotoxic activity of this sort is usually observed in most of the assays with positive results. Since ETU does not appear very potent and is not extremely toxic to test cells and organisms, it is not surprising to find that ETU does not produce consistent effects in many of the assays reviewed. Consequently, in many instances, mixed results for the same assay type are reported by different investigators, but as reviewed herein, these results may be dependent upon the test conditions in each individual laboratory. A primary shortcoming with many of the reported negative results is that the concentrations or doses used are not high enough for an adequate test for ETU activity. There are also problems with many of the negative assays generally in protocol or reporting, particularly with the in vivo studies (e.g. inappropriate sample number and/or sampling times; inadequate top dose employed).Overall, while ETU does not appear to be a potent genotoxic agent, it is capable of producing genotoxic effects (e.g. gene mutations, structural chromosomal aberrations). This provides a basis for weak genotoxic activity by ETU. Furthermore, based on a suggestive dominant lethal positive result, there may be a concern for heritable effects. Due to the many problems with the conduct and assessment of the in vivo assays, it is worth repeating in vivo     

Cloning, purification, and biochemical characterization of the pneumococcal bacteriophage Cp-1 lysin.

Cp-1, a small virulent bacteriophage infecting Streptococcus pneumoniae, encodes its own lytic enzyme (CPL). A fragment of Cp-1 DNA containing the gene cpl coding for CPL was cloned and expressed in high amounts in Escherichia coli. CPL was purified to electrophoretic homogeneity by using affinity chromatography on choline-Sepharose (T. Briese and R. Hakenbeck, Eur. J. Biochem. 146:417-427, 1985), and the enzyme showing a Mr of 39,000 was characterized as a muramidase. This muramidase required for in vivo and in vitro activity the presence of choline in the teichoic acids of the pneumococcal cell walls. Free choline or lipoteichoic acid noncompetitively inhibited the activity of CPL.     

Structural and dynamic properties of bacterial type IV secretion systems (review)

The type IV secretion systems (T4SS) are widely distributed among the gram-negative and -positive bacteria. These systems mediate the transfer of DNA and protein substrates across the cell envelope to bacterial or eukaryotic cells generally through a process requiring direct cell-to-cell contact. Bacteria have evolved T4SS for survival during establishment of pathogenic or symbiotic relationships with eukaryotic hosts. The Agrobacterium tumefaciens VirB/D4 T4SS and related conjugation machines serve as models for detailed mechanistic studies aimed at elucidating the nature of translocation signals, machine assembly pathways and architectures, and the dynamics of substrate translocation. The A. tumefaciens VirB/D4 T4SS are polar-localized organelles composed of a secretion channel and an extracellular T pilus. These T4SS are assembled from 11 or more subunits. whose membrane topologies, intersubunit contacts and, in some cases, 3-dimensional structures are known. Recently, powerful in vivo assays have identified C-terminal translocation signals, defined for the first time the translocation route for a DNA substrate through a type IV secretion channel, and supplied evidence that ATP energy consumption contributes to a late stage of machine morphogenesis. Together, these recent findings describe the mechanics of type IV secretion in unprecedented detail.     

Drosophila basement membrane procollagen alpha 1(IV). II. Complete cDNA sequence, genomic structure, and general implications for supramolecular assemblies

A Drosophila melanogaster gene for a basement membrane procollagen chain was recently identified from the sequence homology of the carboxyl (NC1) end of the polypeptide that it encodes with the corresponding domain of human and murine collagens IV (Blumberg, B., MacKrell, A. J., Olson, P. F., Kurkinen, M., Monson, J. M., Natzle, J. E., and Fessler, J. H. (1987) J. Biol. Chem. 262, 5947-5950). This gene is at chromosome location 25C. Here we report the complete 6-kilobase cDNA sequence coding for a chain of 1775 amino acids, as well as the genomic structure. The gene is composed of nine relatively large exons separated by eight relatively small introns. This organization is different from the multiple small exons separated by large introns reported for mouse and human type IV collagens (Kurkinen, M., Bernard, M. P., Barlow, D. P., and Chow, L. T. (1985) Nature 317, 177-179. Sakurai, Y., Sullivan, M., and Yamada, Y. (1986) J. Biol. Chem. 261, 6654-6657. Soininen, R., Tikka, L., Chow, L., Pihlajaniemi, T., Kurkinen, M., Prockop, D. J., Boyd, C. D., and Tryggvason, K. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1568-1572). Drosophila and human alpha 1(IV) procollagen chains share not only polypeptide domains near their amino and carboxyl ends for making specialized, intermolecular junctional complexes, but also 11 of 21 sites of imperfections of the collagen triple helix. However, neither the number nor the nature of the amino acids in these imperfections appear to have been conserved. These imperfections of the helical sequence may be important for the supramolecular assembly of basement membrane collagen. The 9 cysteine residues of the Drosophila collagen thread domain are arranged as several variations of a motif found in vertebrate collagens IV only near their amino ends, in their "7 S" junctional domains. The relative positions of these cysteine residues provide numerous opportunities for disulfide bonding between molecules in both parallel and antiparallel arrays. There is a pseudorepeat of one-third of the thread length, and there are numerous possibilities for disulfide-linked microfibrils and networks. We propose that collagen microfibrils, stabilized by disulfide segment junctions, are a versatile ancestral form from which specialized collagen fibers and networks arose.     

Structural and biochemical characterization of calhepatin, an S100-like calcium-binding protein from the liver of lungfish (Lepidosiren paradoxa).

We report the biochemical characterization of calhepatin, a calcium-binding protein of the S100 family, isolated from lungfish (Lepidosiren paradoxa) liver. The primary structure, determined by Edman degradation and MS/MS, shows that the sequence identities with the other members of the family are lower than those between S100 proteins from different species. Calhepatin is composed of 75 residues and has a molecular mass of 8670 Da. It is smaller than calbindin D(9k) (78 residues), the smallest S100 described so far. Sequence analysis and molecular modelling predict the two EF-hand motifs characteristic of the S100 family. Metal-binding properties were studied by a direct 45Ca2+-binding assay and by fluorescence titration. Calhepatin binds Ca2+ and Cu2+ but not Zn2+. Cu2+ binding does not change the affinity of calhepatin for Ca2+. Calhepatin undergoes a conformational change upon Ca2+ binding as shown by the increase in its intrinsic fluorescence intensity and lambda(max), the decrease in the apo-calhepatin hydrodynamic volume, and the Ca2+-dependent binding of the protein to phenyl-Superose. Like most S100 proteins, calhepatin tends to form noncovalently associated dimers. These data suggest that calhepatin is probably involved in Ca2+-signal transduction.