Enzyme kinetics of a highly purified mitochondrial creatine kinase in comparison with cytosolic forms

Summary Mitochondrial creatine kinase (CK) purified from canine myocardium showed a single protein band on SDS-PAGE and was free of MMCK. Its amino acid composition was different than MMCK or BBCK and did not react to antiserum to MMCK or BBCK. Using purified mitochondrial, MM and BBCK, the velocity of reaction (V) was estimated for creatine phosphate (CP), creatine (C), adenosine triphosphate (ATP) and adenosine diphosphate (ADP) over a wide range of concentrations including those at V_max. The values for K_m (mM/L) derived from Lineweaver-Burke plots are shown: The affinity of mitochondrial CK for C is much greater than MMCK which is compatible with the energy shuttle hypothesis, namely ATP is converted by mitochondrial CK to CP, and then diffuses to the myofibril for conversion to ATP for utilization.     

Restriction endonuclease EcoO109 from Escherichia coli H709c with heptanucleotide recognition site 5'-PuG/GNCCPy

A new restriction endonuclease, EcoO109, has been isolated from Escherichia coli H709c by polyethyleneimine (PEI) precipitation, DEAE-cellulose chromatography and heparin agarose chromatography. The yield was high, more than 3000 units/g of wet cells. The EcoO109 endonuclease recognizes and cleaves a nucleotide sequence of (formula: see text), in the presence of 10 mM Mg2+. The enzyme will be useful for structural analysis and molecular cloning of DNA because of the stability, high yield and easy handling of the producer strain.     

The isolation and restriction mapping of a miniplasmid from the Actinomycete Nocardia corallina

Abstract A variety of plasmids has been identified as covalently closed circular and linear DNA in certain Actinomycetes, such as Streptomyces . This paper describes the first isolation and characterisation of a plasmid from the genus Nocardia . The plasmid pKU100 isolated from Nocardia corallina is a cccDNA molecule, 2.7 kb in length. This plasmid has been mapped with a wide variety of restriction enzymes and contains a number of unique restriction sites making it suitable for development as a cloning vector.     

Screening of plasmids in non-pathogenic corynebacteria

Abstract A screening of plasmids in 25 nonpathogenic coryneform bacteria was carried out. 11 Strains showed at least one plasmid, ranging in size from 4.2 to 55 kb. These plasmids did not encode bacteriocin production or resistance to a number of antibiotics or to ions such as arsenite, mercury(II) and cobalt(II). A detailed study of plasmid pBL100 from Brevibacterium linens is presented. pBL100 has a size of 7.75 kb, and contains single sites for the endonucleases: Hin dIII; Pst I, Bgl II, Eco RI and Bam HI. B. linens is easily and efficiently transformed with vectors derived from pBL1 isolated from Brevibacterium lactofermentum .     

Alterations in the number of rRNA operons within the Bacillus subtilis genome

Deletions and additions of rRNA gene sets in Bacillus subtilis were observed by Southern hybridizations using cloned radiolabeled rDNA sequences. Of the ten rRNA gene sets found in B. subtilis 168M or NCTC3610, one was deleted in strains possessing the leuB1, ilvC1, argA2 and pheA1 mutations. Among EcoRI restriction fragments of genomic DNA products, a 2.9-kb 23S rRNA homolog was missing. In HindIII digest, both 5.5- and 5.1-kb hybrid bands were lost with 16S and 23S probes, respectively. Similarly, genomic DNAs digested with SmaI showed the absence of both 2.1- and 2.0-kb fragments that hybridized to 16S and 5S sequences, respectively, in wild-type genomes. In contrast, B. subtilis strain 166 and its derivatives displayed a gain of a 3.3-kb HindIII fragment homologous to 16S rRNA. Transforming the ilvC1 and leuB1 mutations into new genetic backgrounds revealed in some clones the concomitant introduction of the ribosomal defect. Transformations with the slightly heterologous donor DNA from strain W23 yielded some Leu+ and Arg+ transformants with altered hybridization patterns when probed with cloned sequences. We propose that the deletion of the rRNA operon occurred in the ilv-leu gene cluster of the B. subtilis genome as a result of unequal recombination between redundant sequences.     

中国人ε珠蛋白基因5′侧序列中多态性Hinc Ⅱ位点的进一步研究

本文进一步研究了我国不同民族的正常个体以及β地中海贫血患者θ珠蛋白基因5′侧序列中的多态性HincⅡ位点及其遗传性质。在广西壮族正常个体和β地中海贫血纯合子中,该多态性位点的发生频率均为75％,与正常汉族人测得值相近。家系分析资料表明,该多态性位点完全按照孟德尔规律进行遗传。     

Leucine biosynthesis in Alcaligenes eutrophus H16: Influence of amino acid additions on the formation of active α-isopropylmalate synthase and α-acetohydroxy acid synthase

The -isopropylmalate synthase of the chemolithoautotrophic Alcaligenes eutrophus H16 is apparently a soluble enzyme but is strongly adsorbed to cell particles in ruptured cell suspensions. This was not observed with -acetohydroxy acid synthase or threonine deaminase. The formation of these regulatory enzymes of the branched chain amino acid biosynthesis pathway generally decreased with decreased growth rates. The addition of 5 mM valine plus isoleucine with and without 5 mM threonine caused a 6.6- and a 4-fold increase, respectively, in the formation of active -isopropylmalate synthase, but caused a strong decrease in the -actohydroxy acid synthase. The level of active -isopropylmalate synthase is apparently regulated by the level of leucine; whereas, the level of the -acetohydroxy acid synthase and threonine deaminase is influenced by the presence of several amino acids. A catabolic threonine deaminase was not encountered.Abbreviations IRS -Isopropylamalate - AHA -acetohydroxy acid - TDA throninedeaminase This paper is dedicated to Professor H. G. Schlegel, University Göttingen, on the occasion of his 60th birthday. I am grateful to a great teacher and scientist, who in his unique way stimulated enthusiasm and fascination in microbiology in his students throughout the years     

Alterations in the structure of the electrical double layer adjacent to a charged membrane arising from electromagnetically induced changes in the activity of membrane bound enzymes

Electromagnetic fields of very low amplitude have been reported to influence a number of cellular functions. Many of these effects have a high degree of frequency specificity. Herein it is suggested that some of these reported results could be explained by a fieldinduced alteration in the enzymic activity of integral membrane proteins. It is shown that such a field-induced transition from an initial nonequilibrium steady-state to a final nonequilibrium steady-state can lead to an alteration in the concentration profiles of those charged species in the cell's ambient electrolyte that comprise the so-called electrical double layer. Examples of variations in the concentration profiles of those ions that react with a membrane-bound enzyme, as well as nonreacting ionic species, are given. The modulation of such effects by systematic variations in extracellular pH and ionic strength is discussed.     

Glutathione-S-transferase,a possible drug-metabolizing enzyme,in Haemonchus contortus: Comparative activity of a cambendazole-resistant and a susceptible strain

Kawalek J. C., Rew R. S. and Heavner J. 1984. Glutathione-S-transferase, a possible drug-metabolizing enzyme in Haemonchus contortus: comparative activity of a cambendazole-resistant and a susceptible strain. International Journal for Parasitology14: 173–175. A drug metabolizing enzyme (DME), glutathione-S-transferase, was detected in homogenates of a cambendazole-susceptible and a cambendazole-resistant strain of Haemonchus contortus. The activity was 1.5–1.8 times higher in the resistant strain. DME activation is a possible mechanism for anthelmintic resistance in H. contortus.