Purification, characterization, and molecular cloning of a 60-kDa phosphoprotein in rabbit skeletal sarcoplasmic reticulum which is an isoform of phosphoglucomutase.

A 60-kDa substrate of calmodulin-dependent protein kinase in rabbit "heavy" skeletal sarcoplasmic reticulum (SR) was characterized by purification and cDNA cloning. Purification was achieved by column chromatography using DEAE-Sephacel, heparin-agarose, and hydroxylapatite in 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid (CHAPS). Analyses of amino acid sequence and composition indicated that the CHAPS-soluble 60-kDa protein is an isoform of phosphoglucomutase (PGM). cDNAs encoding two isoforms of PGM were isolated from rabbit skeletal muscles. The translated amino acid sequences show that the isoforms, PGM1 and PGM2, differ in the N-terminal 77 amino acids and that PGM2 is identical to the 60-kDa protein in the SR. Northern blot analysis showed that the size of the mRNA encoding PGM2 is 2.4 kilobases. The PGM enzyme activity was markedly inhibited in SR membranes, while perturbation of the membranes with CHAPS or guanidine-HCl recovered the enzyme activity. KCl (0.15-1 M) led to a partial recovery of the enzyme activity suggesting that the charge interaction is not the primary force for PGM-SR interaction. PGM is localized in the heavy fraction of SR, where calsequestrin and Ca2+ release channel are enriched. Our results demonstrate that an isoform of PGM localized in junctional skeletal SR is the 60-kDa substrate of calmodulin-dependent protein kinase.     

Analysis of the importance of arginine 102 in neutral endopeptidase (enkephalinase) catalysis.

Neutral endopeptidase 24.11 contains an active site arginine believed to function in substrate binding. This arginine is thought to form an ionic interaction with the COOH-terminal carboxylate of NEP substrates. The functionality of arginine 102 has been investigated by using site-directed mutagenesis to produce mutants in which this residue was converted to a lysine, glycine, glutamine, or glutamate. All of the mutants exhibited essentially full activity as determined with a synthetic peptide amide, glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide. In contrast, activity was detected only with the wild-type enzyme and the lysine mutant using a synthetic substrate containing a free COOH-terminal carboxylate, dansyl-Gly-Trp-Gly. Inhibition studies with the physiologically active peptide substrates substance P, endothelin, and angiotensin I, as well as substance P free acid, [D-Ala2,Leu5]enkephalin, and [D-Ala2,Leu5]enkephalinamide indicated a lack of importance of arginine 102 in substrate binding. With [D-Ala2,Met5]enkephalin and the chemotactic peptide, N-formyl-Met-Leu-Phe, a significant decrease in affinity is observed with the arginine 102 mutants. These results suggest that the contribution of arginine 102 to substrate binding is dependent upon the strength of other subsite interactions. Examination of dipeptides as inhibitors indicates that the nature and orientation of the P'2 residue is important in determining the strength of the interaction of arginine 102 with its substrates.     

Identification of expression signals of the mycobacteriophages Bxb1, L1 and TM4 using the Escherichia-Mycobacterium shuttle plasmids pYUB75 and pYUB76 designed to create translational fusions to the lacZ gene.

Mycobacterial expression signals were cloned using specially constructed gene fusion shuttle plasmid probes carrying a truncated Escherichia coli lacZ (beta-galactosidase) gene which lacked a promoter, a ribosome binding site, and an ATG start codon. Libraries of mycobacteriophage Bxb1, L1 and TM4 DNAs were constructed, and introduced by electroporation into Mycobacterium smegmatis and the 'bacille Calmette-Guérin' (BCG). Clones carrying mycobacterial expression sequences were detected by their blue colour or characteristic fluorescence when plated on media containing chromogenic or fluorogenic substrates. Varying degrees of beta-galactosidase expression were observed, and one Bxb1 expression signal was identified where beta-galactosidase expression is repressed in phage lysogens.     

Predicting protein secondary structure content. A tandem neural network approach.

A priori knowledge of secondary structure content can be of great use in theoretical and experimental determination of protein structure. We present a method that uses two computer-simulated neural networks placed in "tandem" to predict the secondary structure content of water-soluble, globular proteins. The first of the two networks, NET1, predicts a protein's helix and strand content given information about the protein's amino acid composition, molecular weight and heme presence. Because NET1 contained more adjustable parameters (network weights) than learning examples, this network experienced problems with memorization, which is the inability to generalize onto new, never-seen-before examples. To overcome this problem, we designed a second network, NET2, which learned to determine when NET1 was in a state of generalization. Together, these two networks produce prediction errors as low as 5.0% and 5.6% for helix and strand content, respectively, on a set of protein crystal structures bearing little homology to those used in network training. A comparison between three other methods including a multiple linear regression analysis, a non-hidden-node network analysis and a secondary structure assignment analysis reveals that our tandem neural network scheme is, indeed, the best method for predicting secondary structure content. The results of our analysis suggest that the knowledge of sequence information is not necessary for highly accurate predictions of protein secondary structure content.     

Immunohistochemical localization of 36 and 29 kDa proteins in sparganum.

Antigenic proteins of 36 and 29 kDa were localized in Spirometra mansoni plerocercoid (sparganum) immunohistochemically by avidin biotin complex (ABC) staining. When polyclonal antibodies such as BALB/c mouse serum immunized with crude saline extract of sparganum or confirmed sparganosis sera were reacted as primary antibodies, the positive chromogen (3-amino, 9-ethylcarbazole) reactions were recognized at syncytial tegument, tegumental cells, muscle and parenchymal cells and lining cells of excretory canals. A monoclonal antibody (MAb) which was reacting to 36 and 29 kDa proteins in the extract of the worm was localized at the syncytial tegument and tegumental cells. The present results suggested that the potent antigenic proteins of 36 and 29 kDa in sparganum were produced at the tegumental cells and transported to the syncytial tegument.     

Karyotypes of Pneumocystis carinii from Korean rats.

Molecular karyotyping was applied to Pneumocystis carinii(Pc) from two strains of experimental rats, Sprague Dawley(SD) and Fisher(F), in Korea. Field inversion gel electrophoresis and contour clamped homogeneous electric field electrophoresis resolved 15 chromosomal bands from the Pc. The size of the bands was estimated 270kb to 684kb from SD rats, and 273kb to 713 kb from F rats. The bands of 283 kb from SD rats and of 273 kb from F rats stained more brightly suggesting duplicated bands. Total number of chromosomes was at least 16, and total genomic size was estimated 7 x 10(6) bp. All of the bands from F rats hybridized to the probe of repeated DNA sequences of Pc and the band of 448 kb size was proved to contain rDNA sequences, but Pc. chromosome bands from SD rats showed no reactions to the probes. The 2 different karyotypes of P. carinii from 2 strains of rats were maintained consistently for 2 years.     

Anticlastogenic effect of flavonoids against mutagen-induced micronuclei in mice.

14 flavonoids, including flavone and flavonol derivatives, were tested for their anticlastogenic effect against induction of micronuclei by benzo[a]pyrene in polychromatic erythrocytes of mice. When each flavonoid was administered orally, together with intraperitoneally administered benzo[a]pyrene, most flavonol derivatives showed an anticlastogenic effect. The data suggest that the 2,3-double bond and 3,5,7-hydroxyl groups in the flavonoid molecules may be essential to produce anticlastogenic effects against benzo[a]pyrene. Galangin, one of the active compounds, and (-)-epicatechin, a weak one, were administered to mice in order to compare their anticlastogenic effect against 3 different kinds of carcinogens: ethyl methanesulfonate, 7,12-dimethylbenz[a]anthracene, and adriamycin. Galangin showed a stronger anticlastogenic effect than (-)-epicatechin against ethyl methanesulfonate and 7,12-dimethylbenz[a]anthracene. However, there was no significant effect against adriamycin-induced micronuclei by both compounds. Our study indicates that most flavonoids are anticlastogenic agents. Their anticlastogenic effects are apparently independent of their own clastogenic activities. Furthermore, their anticlastogenic activities do not apply universally to all types of genotoxic chemicals.     

Changes in Esterification of the Uronic Acid Groups of Cell Wall Polysaccharides during Elongation of Maize Coleoptiles

Cell walls of grasses have two major polysaccharides that contain uronic acids, the hemicellulosic glucuronoarabinoxylans and the galactosyluronic acid-rich pectins. A technique whereby esterified uronic acid carboxyl groups are reduced selectively to yield their respective 6,6-dideuterio neutral sugars was used to determine the extent of esterification and changes in esterification of these two uronic acids during elongation of maize (Zea mays L.) coleoptiles. The glucosyluronic acids of glucuronoarabinoxylans did not appear to be esterified at any time during coleoptile elongation. The galactosyluronic acids of embryonal coleoptiles were about 65% esterified, but this proportion increased to nearly 80% during the rapid elongation phase before returning to about 60% at the end of elongation. Methyl esters accounted for about two-thirds of the total esterified galacturonic acid in cell walls of unexpanded coleoptiles. The proportion of methyl esters decreased throughout elongation and did not account for the increase in the proportion of esterified galactosyluronic acid units during growth. The results indicate that the galactosyluronic acid units of grass pectic polysaccharides may be converted to other kinds of esters or form ester-like chemical interactions during expansion of the cell wall. Accumulation of novel esters or ester-like interactions is coincident with covalent attachment of polymers containing galactosyluronic acid units to the cell wall.