Purification and properties of a specific primase-stimulating factor of bovine thymus

The DNA replicase activity of the complex between bovine thymus DNA polymerase alpha and RNA primase was markedly decreased after the purification by ssDNA-cellulose column chromatography. In an attempt to restore the activity by supplementing some fractions eliminated from the purified enzyme, we found that a fraction eluted from the column by increasing salt concentration and 30% ammonium sulfate precipitates of the phosphocellulose-step enzyme possessed a high ability to restore the replicase activity. Thus, the factors were purified to near homogeneity from the two sources and the properties were examined. Both factors were heat-labile and trypsin-sensitive, possessed a native molecular mass of approximately 150-200 kDa as judged by Sephacryl S-200 column chromatography, and were composed of two polypeptides of 146 kDa and 47 kDa on SDS/polyacrylamide gel electrophoresis, indicating that they were an identical protein. The factor, which did not show any DNA polymerase or primase activities by itself, stimulated approximately 20-fold the replicase activity of purified DNA-polymerase-alpha-primase at a very low concentration (10 ng/50 microliter). The factor did not affect the deoxyribonucleotide polymerizing activity of the enzyme complex at all, but specifically stimulated the primase activity only. Thus, we designated the factor as primase-stimulating factor. Although varying the template concentration did not significantly affect the mode of stimulation, increasing the concentration of substrate for primer synthesis (ATP) markedly decreased the extent of stimulation. Thus, the stimulating factor seems to decrease the substrate concentration required for the primase reaction as well as increasing threefold the maximum activity attained by varying the substrate concentration. So far, no ATPase activity has been detected in the factor.     

Gelatinases of metastatic cell lines of murine colonic carcinoma as detected by substrate-gel electrophoresis

Gelatinases were detected in the conditioned medium of murine colonic carcinoma cells by SDS-polyacrylamide gel electrophoresis using gels copolymerized with gelatin. Several gelatinase activities differing in molecular weight were detected but the major activities migrated with molecular weights of 60,000 and 95,000. The enzymes did not hydrolyze bovine serum albumin or casein, and required calcium for activity. All of the gelatinase activities were inhibited by EDTA, 1,10-phenanthroline and dithiothreitol but not by N-ethylmaleimide and phenylmethylsulfonyl fluoride. The 95,000 dalton gelatinase was separated from the 60,000 dalton gelatinase by affinity chromatography on Ricinus communis agglutinin-agarose, and the former activity was markedly increased in highly metastatic cell lines as compared with its activity in poorly metastatic cell lines.     

Maternal inheritance of deleted mitochondrial DNA in a family with mitochondrial myopathy

Skeletal muscles from a mother and her daughter both with chronic progressive ophthalmoplegia were analyzed. Histological and biochemical analyses of their muscle samples showed typical features of this type of mitochondrial myopathy. Southern blot analysis revealed that, in both patients, there were two species of mitochondrial DNA (mtDNA): normal one and partially deleted one. The sizes of the deletion were different; the mutant mtDNAs from the mother and the daughter had about 2.5- and 5-kilobase deletions, respectively. The two mutant mtDNAs shared a common deleted region of 1.2-kilobase. However, both the start and the end of deletion were different between them, implying a novel mode of inheritance. This is the first report that the mutant mtDNA is responsible for the maternal inheritance of a human disease.     

Thermostable alanine racemase from Bacillus stearothermophilus: DNA and protein sequence determination and secondary structure prediction

The nucleotide sequence of the alanine racemase (EC 5.1.1.1) gene from a thermophile, Bacillus stearothermophilus, was determined by the dideoxy chain termination method with universal and synthetic site-specific primers. The amino acid sequence of the enzyme predicted from the nucleotide sequence was confirmed by peptide sequence information derived from the N-terminal amino acid residues and several tryptic fragments. The alanine racemase gene consists of 1158 base pairs encoding a protein of 386 amino acid residues; the molecular weight of the apoenzyme is estimated as 43,341. The racemase gene of B. stearothermophilus has a closely similar size (1158 vs 1167 base pairs) to that of the gene of a mesophile, B. subtilis, but shows a higher preference for codons ending in G or C. A comparison of the amino acid sequence with those of Bacillus subtilis and Salmonella typhimurium dadB and alr enzymes revealed overall sequence homologies of 31-54%, including an identical octapeptide bearing the pyridoxal 5'-phosphate binding site. Although the residues common in the four racemases are not continuously arrayed, these constitute distinct domains and their hydropathy profiles are very similar. The secondary structure of B. stearothermophilus alanine racemase was predicted from the results obtained by theoretical analysis and circular dichroism measurement.     

Gene cloning and sequence determination of leucine dehydrogenase from Bacillus stearothermophilus and structural comparison with other NAD(P)+-dependent dehydrogenases

The gene for leucine dehydrogenase (EC 1.4.1.9) from Bacillus stearothermophilus was cloned and expressed in Escherichia coli. The selection for the cloned gene was based upon activity staining of the replica printed E. coli cells. A transformant showing high leucine dehydrogenase activity was found to carry an about 9 kilobase pair plasmid, which contained 4.6 kilobase pairs of B. stearothermophilus DNA. The nucleotide sequence including the 1287 base pair coding region of the leucine dehydrogenase gene was determined by the dideoxy chain termination method. The translated amino acid sequence was confirmed by automated Edman degradation of several peptide fragments produced from the purified enzyme by trypsin digestion. The polypeptide contained 429 amino acid residues corresponding to the subunit (Mr 49,000) of the hexameric enzyme. Comparison of the amino acid sequence of leucine dehydrogenase with those of other pyridine nucleotide dependent oxidoreductases registered in a protein data bank revealed significant sequence similarity, particularly between leucine and glutamate dehydrogenases, in the regions containing the coenzyme binding domain and certain specific residues with catalytic importance.     

Nuclear magnetic resonance studies of cis-syn, trans-syn, and 6-4 photodimers of thymidylyl(3'-5')thymidine monophosphate and cis-syn photodimers of thymidylyl(3'-5')thymidine cyanoethyl phosphotriester

Three out of four possible photodimers of thymidylyl(3'-5')thymidine monophosphates (i.e., cis-syn, 6-4, and one of the trans-syn) and two structural isomers (i.e., R and S forms) of cis-syn-thymidylyl(3'-5')thymidine cyanoethyl phosphotriester have been isolated and purified from the reaction mixtures after UV irradiation and studied by multinuclear magnetic resonance Spectroscopy. All five inter thymine base linked photodimers have grossly similar structures which are quite different from those of the parent thymidylyl(3'-5')thymidine. The base of Tp- is in the syn conformation, and that of -pT it is in the anti conformation. The sugar puckering of Tp- is dominated by the 2E conformer, but in -pT it is in 4E; except for the conformer around C5'-O5' bond, the 6-4 isomer is very similar to those of cis-syn and trans-syn conformation. As expected, there are sugar-phosphate backbone distortions in the phosphotriesters, due to the neutralization of the negative charge of the phosphate. In general the structures of all five photodimers are very close to those of the cis-syn photodimer of thymidylyl(3'-5')thymidine monophosphate cyanoethyl ester as studied by X-ray diffraction [Cadet, J., Voituriez, L., Hruska, F. E., & Grand, A. (1985) Biopolymers 24, 897-903; Hruska, F. E., Voituriez, L., Grand, A., & Cadet, J. (1986) Biopolymers 25, 1401-1417]. While the trans-syn photodimer has two structural isomers, only one [C6(of Tp-)-R] was produced by the UV irradiation and studied.     

The plasma binding protein for vitamin D is a site of discrimination against vitamin D-2 compounds by the chick

The binding of 25-hydroxy-[26,27-3H]vitamin D-3 and 25-hydroxy-[26,27-3H]vitamin D-2 to the vitamin D binding protein in the plasma of both rats and chicks has been studied. In the case of rats, sucrose density gradient analysis, competitive displacement, and Scatchard analysis demonstrate that 25-hydroxyvitamin D-3 and 25-hydroxyvitamin D-2 are bound equally well to the vitamin D binding protein. In contrast, 25-hydroxyvitamin D-2 is poorly bound, while 25-hydroxyvitamin D-3 is tightly bound to the vitamin D binding protein in chick plasma. On the other hand, the chick intestinal receptor binds 1,25-dihydroxyvitamin D-2 and 1,25-dihydroxyvitamin D-3 equally well with a KD of 7.10(-11) M for both compounds. These results strongly suggest that the failure of the plasma transport protein in chicks to bind the vitamin D-2 compounds may be responsible for their relative ineffectiveness in these animals.     

Molecular cloning and nucleotide sequence of Thermus thermophilus HB8 trpE and trpG

The trpE gene of Thermus thermophilus HB8 was cloned by complementation of an Escherichia coli tryptophan auxotroph. The E. coli harboring the cloned gene produced the anthranilate synthase I, which was heat-stable and enzymatically active at higher temperature. The nucleotide sequence of the trpE gene and its flanking regions was determined. The trpE gene was preceded by an attenuator-like structure and followed by the trpG gene, with a short gap between them. No other gene essential for tryptophan biosynthesis was observed after the trpG gene. The amino-acid sequences of the T. themophilus anthranilate synthase I and II deduced from the nucleotide sequence were compared with those of other organisms.     

Use of polyelectrolyte complex-stabilized calcium alginate gel for entrapment of beta-amylase

Calcium alginate gel stabilized with a polyelectrolyte complex (PEC) consisting of potassium poly(vinyl alcohol) sulfate (KPVS) and trimethylammonium glycol chitosan iodide (TGCI) was used for the immobilization of beta-amylase. The immobilization was made by gelling aqueous droplets of enzyme solution including both sodium alginate and KPVS in a CaCl(2) solution containing TGCI. The activity of the enzyme entrapped into the stabilized gel beads was evaluated by studying the batch reaction kinetics of enzyme-catalyzed hydrolysis of maltotetraose. Repeated kinetic measurements, totaling 18, were carried out at fixed time intervals. After each measurement the beads were stirred for 1 day in a freshly prepared 10 mM NaCl solution at 3 degrees C. It was found that the immobilized system remained stable without leading to a serious loss of the activity or to a large leakage of the enzyme from the support. This was explained as being due to a PEC-crosslinked contracted network structure of the stabilized gel matrix.