Reconstitution of active octameric mitochondrial creatine kinase from two genetically engineered fragments.

Creatine kinase (CK) has been postulated to consist of two flexibly hinged domains. A previously demonstrated protease-sensitive site in M-CK (Morris & Jackson, 1991) has directed our attempts to dissect mitochondrial CK (Mi-CK) into two protein fragments encompassing amino acids [1-167] and [168-380]. When expressed separately in Escherichia coli, the two fragments yielded large amounts of insoluble inclusion bodies, from which the respective polypeptides could be purified by a simple two-step procedure.

Autophosphorylation of creatine kinase: characterization and identification of a specifically phosphorylated peptide.

We report that several different chicken and rabbit creatine kinase (CK)1 isoenzymes showed an incorporation of 32P when incubated with [gamma-32P]ATP in an autophosphorylation assay. This modification was was shown to be of covalent nature and resulted from an intramolecular phosphorylation reaction that was not dependent on the CK enzymatic activity. By limited proteolysis and sequence analysis of the resulting peptides, the autophosphorylation sites of chicken brain-type CK could be localized within the primary sequence of the enzyme to a 4.

Role of a small RNA pol II subunit in TATA to transcription start site spacing.

The yeast shi mutation affects the spacing between the TATA promoter element and transcription initiation sites; for the H2B and ADH1 genes, a series of start sites located approximately 50-80 bp downstream of TATA is used in addition to the wild-type initiation sites located at around 100 bp from TATA (1). Here, the yeast SHI wild-type gene has been isolated by complementation and shown to be identical to RPB9, the gene encoding a small subunit of RNA polymerase II. A point mutation in the shi gene, changing a cysteine residue in a putative zinc ribbon motif into a phenylalanine residue, was demonstrated to permit the observed usage of upstream initiation sites.

The tryptophan residues of mitochondrial creatine kinase: roles of Trp-223, Trp-206, and Trp-264 in active-site and quaternary structure formation.

The 5 tryptophan residues of chicken sarcomeric mitochondrial creatine kinase (Mib-CK) were individually replaced by phenylalanine or cysteine using site-directed mutagenesis. The mutant proteins were analyzed by enzyme kinetics, fluorescence spectroscopy, circular dichroism, and conformational stability studies. In the present work, Trp-223 is identified as an active-site residue whose replacement even by phenylalanine resulted in > or = 96% inactivation of the enzyme.

Creatine kinase isoenzymes in chicken cerebellum: specific localization of brain-type creatine kinase in Bergmann glial cells and muscle-type creatine kinase in Purkinje neurons.

Creatine kinase isoenzymes were localized in the chicken cerebellum by the use of isoenzyme-specific anti-chicken creatine kinase antibodies. Brain-type creatine kinase was found in high amounts in the molecular layer, particularly in Bergmann glial cells but also in other cells of the cerebellar cortex, e.g.

Creatine kinase: the reactive cysteine is required for synergism but is nonessential for catalysis.

Chemical modification of rabbit muscle creatine kinase (CK) with thiol-specific reagents led to partial or complete inactivation of the enzyme. Using site-directed mutagenesis, we have substituted the corresponding reactive Cys278 in the chicken cardiac mitochondrial creatine kinase (Mib-CK) with either glycine, serine, alanine, asparagine, or aspartate. The resulting mutant Mib-CK enzymes showed qualitatively similar changes in their enzymatic properties.

Expression of active octameric chicken cardiac mitochondrial creatine kinase in Escherichia coli.

Sarcomeric mitochondrial creatine kinase (Mib-CK) of chicken was expressed in Escherichia coli as a soluble enzyme by using an inducible phage-T7 promoter. Up to one third of the protein in E. coli extracts consisted of soluble recombinant Mib-CK in an enzymically active form.

SHI, a new yeast gene affecting the spacing between TATA and transcription initiation sites.

In a genetic selection for Saccharomyces cerevisiae genes involved in transcription start site specification, two mutant genes which restore alcohol dehydrogenase activity to a functionally defective S. pombe ADH gene were recovered. Examination of S.

DNA sequence elements required for transcription initiation of the Schizosaccharomyces pombe ADH gene in Saccharomyces cerevisiae.

The roles of the TATA element and sequences near the mRNA initiation site in specifying the location of initiation sites in Saccharomyces cerevisiae were examined, using the Schizosaccharomyces pombe ADH gene. The importance of spacing was demonstrated by analysis of a series of deletions that removed from 8-50 bp between the TATA element and ATG translation initiation site of this gene. Primer extension mapping showed that increasing deletion length is associated with a progressive shift downstream in the location of the initiation sites.