Calmodulin fragments can not activate target enzymes

Under conditions where nM level of calmodulin was able to show full activation of myosin light chain kinase and cyclic-nucleotide phosphodiesterase, the fragments of calmodulin at concentrations as high as 20 microM failed to activate these enzymes in the presence of Ca2+. The fragments tested were Ala1-Lys75 (F12), Ala1-Arg74 (F12'), Lys75-Lys148 (F34'), Met76-Lys148 (F34'), Asp78-Lys148 (F34), Ala1-Arg106 (F123), and His107-Lys148 (F4). Purification of the proteolytic fragments through HPLC was necessary to remove contaminant calmodulin. Among the fragments, that corresponding to the C-terminal half domain inhibited myosin light chain kinase activity with the inhibition constant of 13 microM. The integrated structure of calmodulin consisting of N-terminal half domain, C-terminal half domain, and the linker peptide was indispensable for the enzyme activation. We discuss the functions of the two structural domains (N-domain and C-domain) in the activation of various enzymes.     

Models for depolymerizing enzymes. Application to α-amylases

Several research groups have explored the action pattern of depolymerizing enzymes by examining the distribution of products during the early stages of digestion. Mathematical models for the various mechanisms that have been proposed for the behaviour of depolymerizing enzymes are developed in this paper. Published data for several amylases are reexamined in light of this theoretical analysis. It is concluded that following an initial random attack on a long polymer substrate, the enzyme frequently releases only one of the substrate fragments. The retained fragment may be repetitively hydrolyzed near one end to release a series of oligosaccharides before the enzyme substrate–fragment complex finally dissociates. Near optimum conditions Bacillus subtilis (var. amyloliquefaciens) amylase is unique among the enzymes tested because it does not repetitively attack substrate fragments.     

Restriction enzyme cleavage sites surrounding the structural gene for the lipoprotein of the Escherichia coli outer membrane.

The purified messenger ribonucleic acid (mRNA) for the lipoprotein of the Escherichia coli outer membrane was hybridized with fragments obtained by digestion of E. coli chromosomal deoxyribonucleic acid (DNA) with eight different restriction enzymes. For each restriction enzyme digestion, one specific fragment separated by agarose gel electrophoresis was found to hybridize with the lipoprotein mRNA. From the analysis of restriction fragments generated by double digestions with various combinations of restriction enzymes, cleavage sites for the restriction enzymes near the locus of the lipoprotein structural gene (lpp) were mapped. No restriction fragments of DNA from the E. coli lpp-2 mutant hybridized with the lipoprotein mRNA, confirming that the mutant has a deletion mutation in the vicinity of the lpp gene.     

On the differences of molecular aggregates structure with long wave fluorescence in the photosystems I and II]

Galactolipase and protenase action on the low temperature fluorescence spectra of light chloroplast fragments obtained from grana or intergrana thylakoids and grana thylakoids system before and after isolation photosystem I particles have been studied. Identical hydrolytic enzymes action in the two type photosystem I particles have been studied. Identical hydrolytic enzymes action in the two type photosystem I particles were observed. Grana thylakoids system after removing photosystem I particles contained photosystem II in the most purified form. These measurements results confirmed our previous suggestion that the band at 735 nm in the low temperature fluorescence spectra of light and heavy fragments belongs to the different native chlorophyll a aggregates.     

Gene localization, size, and physical map of the chromosome of Streptococcus pneumoniae.

A physical map of the Streptococcus (Diplococcus) pneumoniae chromosome, which is circular and 2,270 kbp in circumference, has been constructed. The restriction enzymes ApaI, SmaI, and SacII were used to digest intact chromosomes, and the fragments were resolved by field inversion gel electrophoresis (FIGE). The digests produced 22, 20, and 29 fragments, respectively. The order of the fragments was deduced from Southern blot hybridization of isolated labeled fragments to separated fragments of the various restriction digests. Genetic markers were correlated with the physical map by transformation of recipient cells with FIGE-isolated DNA fragments derived from genetically marked S. pneumoniae strains. In addition, markers were mapped by the hybridization of cloned genes to FIGE-separated restriction fragments. Six rRNA gene (rrn) clusters were mapped by hybridization to rrn-containing fragments of Haemophilus influenzae.     

Tumorigenic poxviruses: construction of the composite physical map of the Shope fibroma virus genome.

The sites for the restriction enzymes BamHI, Bg/I, HindIII, PstI, PvuII, and SstI on the linear DNA genome of Shope fibroma virus, a tumorigenic poxvirus of rabbits, have been determined by digestions of the cloned BamHI and HindIII restriction fragments and by hybridization of 32P-labeled cloned fragments to Southern blots of Shope fibroma virus DNA cleaved partially or completely with the various enzymes. The linear genome is shown to be 160 kilobases in length and to possess terminal inverted repeat sequences of between 12.2 and 12.5 kilobases extending inwards from the cross-linked DNA telomeres. The fine map of the Shope fibroma virus terminal inverted repeats has been constructed and shown to be distinctly different from that of members of the orthopoxvirus group, such as vaccinia, by the absence of detectable tandemly repeated sequences near the termini and by the lack of detectable sequence homology with vaccinia termini.     

An improved method for the large-scale isolation of chromaffin granules from bovine adrenal medulla

Chromaffin granules essentially free of contamination from mitochondria, lysosomes and fragments of endoplasmic reticulum have been isolated in a large scale from bovine adrenal medulla. The homogeneity was judged by electron microscopy and assays of various ‘marker’ enzymes.     

Expression of Ti-plasmid DNA in E. coli: Comparison of homologous fragments cloned from Ti plasmids of Agrobacterium strains C58 and Ach5

Fragments of two Ti plasmids from Agrobacterium tumefaciens were cloned and studied for cross-hybridization. Homologous fragments were further investigated for areas of strong homology and mapped with various restriction enzymes. The fragments were inserted in two directions and the recombinant plasmid was brought into E. coli strains producing minicells. About five proteins were found to be coded by those fragments of the Ti plasmids.     

Organization of the yeast ribosomal RNA gene cluster via cloning and restriction analysis.

The restriction endonuclease EcoR1 cleaves Saccharomyces cerevisiae DNA, which codes for ribosomal RNA (rRNA), into seven fragments, A second restriction endonuclease, HindIII, cleaves the same yeast ribosomal DNA into two fragments. These two restriction enzymes each yield DNA segments that total about 5.9 megadaltons. The "repeat unit" of the yeast genes coding for rRNA is thus about 5.9 megadaltons or about 9000 base pairs long. The two HindIII-cleaved DNA fragments as well as one of the EcoR1-cleaved DNA fragments were purified and amplified by cloning in Escherichia coli. Three of the seven EcoR1-generated DNA fragments could then be ordered by treating the two cloned HindIII DNA fragments with EcoR1. This led the assignment of the two HindIII restriction sites. The various restriction DNA fragments were hybridized directly from the gel utilizing 32P-labeled 5 S, 5.8 S, 18 S, and 25 S rRNA. Identification of the various DNA restriction segments then led to the final ordering of the DNA fragments. The gene coding for the 5 S RNA is adjacent to the gene coding for the 35 S precursor rRNA. These two groups of genes thus occur as a cluster in the following sequence: [5 S-spacer]-[spacer-18 S-5.8 S-25 S-spacer]-[spacer-5 S]. The actual map of the DNA restriction fragments is presented.     

Physical map of the Methanobacterium thermoautotrophicum Marburg chromosome.

A physical map of the Methanobacterium thermoautotrophicum Marburg chromosome was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by NotI, PmeI, and NheI. The order of the fragments was deduced from Southern blot hybridization of NotI fragment probes to various restriction digests and from partial digests. The derived map is circular, and the genome size was estimated to be 1,623 kb. Several cloned genes were hybridized to restriction fragments to locate their positions on the map. Genes coding for proteins involved in the methanogenic pathway were located on the same segment of the circular chromosome. In addition, the genomes of a variety of thermophilic Methanobacterium strains were treated with restriction enzymes and analyzed by pulsed-field gel electrophoresis. The sums of the fragment sizes varied from 1,600 to 1,728 kb among the strains, and widely different macrorestriction patterns were observed.     

Fingerprinting of bacterial genomes by amplification of DNA fragments surrounding rare restriction sites.

A method for generating limited representations of total bacterial DNA, without prior knowledge of the DNA sequence, has been developed. This method consists of three steps: digestion with two restriction enzymes, ligation of two oligonucleotide adapters corresponding to the restriction sites, and selective PCR amplification of the ligation products. The method relies on the use of two restriction enzymes with considerable differences in cleavage frequency of the investigated DNA and the ligation of two different oligonucleotides, each corresponding to one of the two cohesive ends of DNA fragments. Three subsets of DNA fragments are generated during digestion and subsequent ligation: terminated with the same oligonucleotide on both 5' ends of DNA fragments (two subsets) and terminated with two different oligonucleotides. Suppression PCR allows only the third subset of DNA fragments to be amplified exponentially. The method allows bacterial species strain differentiation on the basis of the different DNA band patterns obtained after electrophoresis in polyacrylamide gels stained with ethidium bromide and visualized in UV light.     

The size and a physical map of the chromosome of Haemophilus parainfluenzae

The physical map of the Haemophilus parainfluenzae chromosome is circular and approx. 2340 kb in circumference. The size of the map was determined by digesting agarose-immobilized chromosomes with the restriction enzymes, NotI (GCGGCCGC), RsrII (CGGATCCG) and ApaI (GGGCCC), and using field-inversion gel electrophoresis to resolve the resulting fragments. The enzymes digest the H. parainfluenzae genome into 7, 10, and 18 fragments, respectively. The map order of the fragments was obtained by using Southern-blot hybridization to establish overlapping regions.     

Developmental changes in the structural organization of the lectin discoidin I detected by limited proteolysis

Digestion of discoidin I with several proteolytic enzymes reveals the existence of structural domains in this lectin. Significative differences have been detected in the pattern of fragments generated by V8 protease on discoidin I of various developmental situations. The changes observed can be related to the presence of various types of tetrameric structures in discoidin I. Together with the presence of different types of isoforms in vegetative vs. differentiated cells, the results presented here suggest the involvement of different structural organizations in discoidin I which can be related to the biological functions of this lectin.     

Mode of extension of the daughter strands in the replication of closed circular mitochondrial DNA in vitro.

1. The properties of nascent DNA in the replicative closed circular mitochondrial DNA were examined in the in vitro system of discontinuous replication, using newborn rat liver mitochondria containing endogeneous DNA templates and enzymes. 2. The nascent DNA associated with the closed circular DNA fraction was found to be of two types; one class consisted of the fragments, and the other of the higher molecular-weight DNAs. Data from pulse and chase experiments indicate that the fragments were initially synthesized and subsequently converted into both heavy and light strands of the higher molecular-weight DNAs in an asymmetrical mode. 3. DNA - DNA hybridization experiments revealed that half of the fragments at least were copies of complementary parts of the parental DNA. 4. Based on the present in vitro data, a tentative structure of the replicating region and its expansion are discussed.     

Isoelectric focusing electrophoresis of lignin.

Isoelectric focusing is introduced as a technique for the analysis of macromolecular lignin. The analysis is performed in a pH gradient from 3.5 to 10. Separated lignin fragments are visualized under uv light or by silver staining. The method can be used to distinguish between differently processed lignin preparations and to identify their components. Even the slight modification resulting from attack by ligninolytic enzymes could be detected.     

Restriction enzyme analysis of the Plasmid ColIb DNA

Summary Plasmid ColIb (61.5 Mdal) was digested with restriction enzymes EcoRI and HindIII. The DNA digestion products were separated by electrophoresis on 1.2% agarose gels. There were identified 22 fragments of ColIb DNA generated by the endonuclease EcoRI and 21 fragments produced by HindIII. Molecular weights of the fragments were estimated. The total molecular weight of the fragments generated by EcoRI was 61.42 Mdal and for HindIII fragments 62.79 Mdal.     

Enzymatic synthesis of tRNA fragments

In the present paper the results of enzymatic synthesis of yeast tRNA1Val fragments have been summarized. It is shown that complex use of nucleolytic enzymes is a convenient and effective method of synthesis of the defined sequence oligoribonucleotides. The consecutive use of different nucleolytic enzymes (ribonucleases with different substrate specificity and polynucleotide phosphorylase) and RNA ligase has permitted to obtain various fragments (or their analogs) of T psi-loop, D-arm, anticodon arm and acceptor stem. Some fragments containing modified nucleosides such as tetranucleotide GpDpCpGp (fragment 15-18), octanucleotide GpUpCpUpApGpDpC (analog of fragment 10-17), nonanucleotide GpTpUpCpGpApUpCpC (analog of T psi-loop), decanucleotide psi pCpUpGpCpUpUpIpApC (analog of fragment 27-36), hexanucleotide CpApCpGpCpA (fragment 36-41) and others were synthesized.     

Lysosomal enzyme phosphorylation. Recognition of a protein-dependent determinant allows specific phosphorylation of oligosaccharides present on lysosomal enzymes

We have investigated the basis for the specific recognition of lysosomal enzymes by UDP-GlcNAc:lysosomal enzyme N-acetylglucosaminylphosphotransferase. This enzyme is responsible for the selective phosphorylation of mannose residues on lysosomal enzymes. Two mammalian lysosomal enzymes, cathepsin D and uteroferrin, and two nonlysosomal glycoproteins were treated with endo-beta-N-acetylglucosaminidase H to remove those high mannose oligosaccharide units which are accessible on the native protein. These proteins were then tested as inhibitors of three different glycosyltransferases. The endo H-treated lysosomal enzymes were shown to be specific inhibitors of the phosphorylation of intact lysosomal enzymes. Proteolytic fragments of cathepsin D, including the entire light chain and heavy chain, did not retain the ability to be recognized by the N-acetylglucosaminylphosphotransferase. These findings indicate that the intact protein portion of lysosomal enzymes contains a specific recognition determinant which leads to high-affinity binding to the N-acetylglucosaminylphosphotransferase. The expression of this determinant appears to be dependent on the conformation of the protein.     

The role of DL and DH degradation products in the reactions of the plasmin hydrolysis of fibrinogen and fibrin]

The mechanism of effect of plasmin hydrolysis degradation products, fragments DL and DH, on fibrinolysis and fibrinogenolysis processes was investigated on the basis of their various structural and functional characteristics. Using electrophoresis of unreduced samples and degradation products concentrations changing kinetics, DH was shown to be the only fragment which possessed an antifibrinolytic effect. Rauleigh's light scattering analysis indicated the ability of fragments DL and DH to co-form with plasminogen reversible equimolar complexes.     

Global fluctuations of the immunoglobulin domains under physiological conditions

Hydrogen-exchange rates of the indole NH proton of a tryptophan residue, buried fully in the interior of each of the constant (CL) and variable (VL) fragments of a type-kappa-immunoglobulin light chain, were studied at various pH values and at 25 degrees C under 1H-nuclear magnetic resonance. The activation energies for the exchange reactions were determined also and compared with those for the unfolding reactions of these fragments induced by guanidine hydrochloride. The pH profiles of the exchange rates of the CL(kappa) and VL(kappa) fragments were very similar to that for a CL (lambda) fragment reported previously. It was found that the CL (kappa) and VL (kappa) fragments as well as the CL (lambda) fragment undergo a global unfolding transition with a conformation very similar to that of the fully unfolded state induced by guanidine hydrochloride even under physiological conditions.