Studies on naturally occurring proteinous inhibitor for transmethylation reactions

An inhibitor for S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases has been purified from rat liver particulate fraction to apparent homogeneity, as judged by high-performance liquid chromatography, two-dimensional paper electrophoresis and isoelectric focusing chromatography. This inhibitor molecule, which is composed of 27 amino acid residues with an additional fluorescent chromophore, is rich in glycine, contains no basic amino acid, and has an isoelectric point (pI) of 3.70. A single absorption peak was observed at 248 nm in acidic as well as in neutral media, while two peaks were detected in alkaline medium at 206 nm and 248 nm. The former peak was found to be quite labile. The fluorescent spectra with excitation peak at 285 nm and emission peak at 358 nm are greatly influenced by the pH, being the highest in alkaline medium. The purified inhibitor inhibits all the AdoMet-dependent methyltransferases examined.     

The phosphate group of 3-phosphoglycerate accounts for conformational changes occurring on binding to 3-phosphoglycerate kinase. Enzyme inhibition and thiol reactivity studies

Steady-state kinetic study of the inhibition of 3-phosphoglycerate kinase reaction by the substrate analogues D-glycerol 3-phosphate, 2-phosphoglycolate, tartronate and malonate revealed competition with respect to 3-phosphoglycerate. D-Glycerate had no detectable inhibitory effect. The data indicate that (a) the phosphate of 3-phosphoglycerate plays an essential role in the formation of its complex with the enzyme and, taking into account the relatively strong binding of 3-phosphoglycerate, (b) the two charged groups of the substrate might cause a synergic interaction with the protein. The carboxyl-lacking D-glycerol 3-phosphate is a non-competitive inhibitor with respect to MgATP, while all the investigated carboxyl-containing inhibitors compete for MgATP binding. The inhibitory analogues of 3-phosphoglycerate reduce the reactivity of both the two fast-reacting and the five slow-reacting thiol groups of the enzyme molecule. In the case of the fast-reacting thiols the effect is specifically associated with the presence of a ligand's phosphate group. Similarly mainly the phosphate-containing nucleotides and analogues slow down significantly the reaction rate of the fast-reacting thiols, while adenosine is less effective and the competitive inhibitor adenine has no effect at all. MgADP has an especially dramatic effect as compared to MgATP, in line with the known X-ray structural data. The fast-reacting thiols are of particular interest, since their reactivity is possibly controlled by ligand-induced conformational changes. This is shown by the similar ligand protection against alkylation irrespective of the reagent's electrostatic charge (iodoacetamide or iodoacetate) and also by the similar substrate-binding properties of carboxamidomethylated and the unmodified enzyme.     

Regulation of nitrile-hydratase synthesis in a Brevibacterium species

The regulation of nitrile-hydratase biosynthese was studied in Brevibacterium sp R 312. Enzyme biosynthesis was not influenced by any carbon and nitrogen source used in the growth medium. It was, however, repressed by amide and amide analogues. Acetamide repressed nitrile-hydratase biosynthesis and induced the wide-spectrum amidase. Therefore, it appeared reasonable to hypothesize a single repressor gene for the nitrile-hydratase/wide-spectrum amidase system.     

Spermine as a modulator of membrane fusion: interactions with acidic phospholipids

The interaction of spermine with acidic phospholipids was investigated for its possible relevance to membrane fusion. Equilibrium dialysis was used to measure the binding of spermine and calcium to large unilamellar vesicles (liposomes) of phosphatidate (PA) or phosphatidylserine (PS). Spermine bound to isolated PA and PS liposomes with intrinsic association constants of approximately 2 and 0.2 M-1, respectively. Above the aggregation threshold of the liposomes, the binding of spermine increased dramatically, especially for PA. The increased binding upon aggregation of PA liposomes was interpreted as evidence for the formation of a new binding complex after aggregation. Spermine enhanced calcium binding to PA, while it inhibited calcium binding to PS, under the same conditions. This difference explained the small effect of spermine on the overall rate of calcium-induced fusion of PS liposomes as opposed to the large effect on PA liposomes. The rate increase could be modeled by a spermine-induced increase in the liposome aggregation rate. The preference for binding of spermine to PA over PS suggested a preference for accessible monoesterified phosphate groups by spermine. This preference was confirmed by the large effects of spermine on aggregation and overall fusion rates of liposomes containing phosphatidylinositol 4,5-diphosphate. The large spermine effects on these liposomes compared with phosphatidate- or phosphatidylinositol-containing liposomes suggested that spermine has a strong specific interaction with phosphatidylinositol 4,5-diphosphate. Clearly, phosphorylation of phosphatidylinositol can lead to a large change in the spermine sensitivity of membrane fusion.     

Fusion of liposomes induced by a cationic protein from the acrosome granule of abalone spermatozoa

Lysin, a protein of Mr 16 000 from the acrosome granule of the abalone, is responsible for the dissolution of the egg vitelline layer. The primary structure of this cationic protein projects some hydrophobic domains in the secondary structure. Lysin was found to associate nonselectively with phospholipid bilayers and cause a spontaneous release of encapsulated carboxyfluorescein in liposomes. The association of lysin with phosphatidylcholine liposomes suggests that there is a hydrophobic interaction between lysin and lipid bilayers. Binding of lysin to phospholipid resulted in the aggregation of phosphatidylserine-containing liposomes, but aggregation was not observed in neutral phosphatidylcholine liposomes. Resonance energy transfer and dequenching of fluorescent 1-palmitoyl-2-cis-parinaroylphosphatidylcholine were both used to determine the fusogenic activity of lysin in aggregated liposomes. Results from both assays are consistent. Lysin-induced fusion was observed in all the phosphatidylserine-containing liposomes, and the general trend of fusion susceptibility was phosphatidylserine/phosphatidylcholine (1:2) approximately equal to phosphatidylserine/phosphatidylcholine/phosphatidylethanolamine (1:1:1) greater than phosphatidylserine/phosphatidylethanolamine (1:2). Cholesterol up to 30% did not affect the intrinsic fusion susceptibility. A hydrophobic penetration by protein molecules and the packing of phospholipid bilayers are used to interpret the fusion susceptibility. Lysin-induced liposome aggregation was highly independent of the state of self-association of lysin in ionic medium. However, the fusogenic activity of self-associated lysin was found to be much less than the monodispersed one. Liposomes preincubated with Ca2+ did not fuse initially as readily as those without Ca2+ treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of a cDNA for human muscle 6-phosphofructokinase

A cDNA for human muscle 6-phosphofructokinase (EC.2.7.1.11) has been isolated from a human fibroblast cDNA library made using the Okayama-Berg procedure. The cDNA isolated as a Bam H1 fragment of the pcD recombinant, pO4, is approximately 2000 bp in length. It represents approximately 1350 bp of the C-terminus coding sequence of the enzyme, approximately 500 bp of the 3'-untranslated region and approximately 150 bp of the vector sequences. The identity of the pO4 cDNA was established by the observation of a high degree of homology (approximately 95%) between the deduced amino acid sequence with the published protein sequence of rabbit muscle 6-phosphofructokinase, and the assignment of the sequence to human chromosome 1 (the known location of PFKM) by using somatic cell hybrids. Based on immunochemical evidence, we had previously predicted not only a remarkable structural conservation of the vertebrate muscle PFK, but also partial structural identity among all three vertebrate PFK isozymes. The pO4 cDNA is, therefore, expected to permit isolation of cDNAs for muscle and non-muscle PFKs from a wide variety of vertebrate species.     

Localization of the human NCAM gene to band q23 of chromosome 11: the third gene coding for a cell interaction molecule mapped to the distal portion of the long arm of chromosome 11

cDNA clones containing sequences coding for the murine neural cell adhesion molecule (N-CAM) were used in Southern hybridizations on human genomic DNA and demonstrated approximately 90% homology between human and murine NCAM genes. In situ hybridization with one of these clones was performed on human metaphase chromosomes and allowed the localization of the human NCAM gene to band q23 of chromosome 11. The genes for two other cell surface molecules believed to be involved in cell-cell interactions, Thy-1 and the delta chain of the T3-T cell receptor complex, have recently been localized to the same region of chromosome 11 in man. Moreover, this region of the human chromosome 11 appears to be syntenic to a region of murine chromosome 9 that also contains the staggerer locus: staggerer mice show abnormal neurological features which may be related to abnormalities in the conversion of the embryonic to the adult forms of the N-CAM molecule.     

UV-induced interstrand cross-linking of d(GT)n.d(CA)n is facilitated by a structural transition

Photochemical alterations following ultraviolet irradiation of the alternating copolymer d(GT)n.d(CA)n were studied. We found that in solution conditions which produced circular dichroism spectra compatible with B-form or A-form DNA, no interstrand cross-linking or photoproduct formation could be demonstrated. Zimmer et al. (Zimmer, C., Tymen, S., Marck, C., and Guschlbaumer, W. (1982) Nucleic Acids Res. 10, 1081-1091) and Vorlickova et al. (Vorlickova, M., Kypr, J., Sotkrova, S., Sponar, J. (1982) Nucleic Acids Res. 10, 1071-1080) have reported a number of solution conditions which produce a structural transition of this polymer characterized by a negative deviation of the circular dichroism spectrum in the region of 280 nm. The nature of this transition has not yet been elucidated. Following ultraviolet irradiation of d(GT)n.d(CA)n under two conditions which produce this transition (manganese solution or ethanol plus trace salts solution) we found ultraviolet dose-dependent interstrand cross-linking as well as dose-dependent formation of thymine-containing photoproduct. Interstrand cross-linking is demonstrated by two criteria: increase in polymer size as detected by alkaline agarose gel electrophoresis, and generation of intermediate density material in alkaline cesium sulfate isopycnic gradients. The thymine-containing photo-product was demonstrated by thin layer chromatography of acid hydrolysates of the polymer. The photo-product is at least partially photoreversible. These findings suggest that the geometry of the alternative conformation is such that pyrimidines from different strands are closely approximated, allowing for photodimerization.     

5,5-Diphenylhydantoin Antagonizes Neurochemical and Behavioral Effects of p, p'-DDT but Not of Chlordecone

Abstract: Rats were given 75 mg/kg of 5,5-diphenylhydantoin (phenytoin) or vehicle 30 min prior to 75 mg/kg of 1, 1, 1-trichloro-bis( p -chlorophenyl)ethane ( p, p' -DDT) (p.o.) or chlordecone (i.p.) and tremor was measured 12 h later. Rats were then killed, and regional brain levels of biogenie amines and their acid metabolites and amino acids were determined. Pretreatment with phenytoin significantly attenuated the tremor produced by p, p' -DDT but enhanced that produced by chlordecone. p, p' -DDT had significant effects on the levels of asparate, glutamate, 5-hydroxyindoleacetic acid (5-HIAA), and 3-methoxy-4-hydroxyphenylglycol (MHPG), whereas chlordecone increased glycine, 5-HIAA, and MHPG levels. Pretreatment with phenytoin blocked p.p' -DDT-induced increases of aspartate in the brainstem and spinal cord, 5-HIAA in the hippocampus, and MHPG in the brainstem and hypothalamus. Phenytoin significantly enhanced chlordecone-induced increases of MHPG in the brainstem. These data indicate that organo-chlorine-induced increases in noradrenergic activity in the brainstem and spinal cord may be directly related to the tremorigenic effects of these chemicals.