High degree of homology between primary structure of human lysosomal acid phosphatase and human prostatic acid phosphatase

Alignment of the amino-acid sequences of the human lysosomal acid phosphatase (LAP) and human prostatic acid phosphatase (PAP) yielded an extensive homology between the two mature polypeptide chains. In the overlapping part, which extends over the entire PAP sequence and the N-terminal 90% of the LAP sequence, the identity is 49.1%. The LAP has an additional C-terminal sequence, which is encoded by the last exon of the LAP gene. This sequence contains the transmembrane domain of LAP, which is lacking in the secretory PAP. All six cysteine residues as well as 20 out of 27 (LAP) and 26 (PAP) proline residues present in the overlapping part of the proteins are conserved, suggesting that they are involved in stabilization of the tertiary structure of both proteins. Only two out of 8 N-glycosylation sites in LAP and 3 in PAP are conserved, suggesting that the dense N-glycosylation of LAP is related to its function in lysosomes.     

Plasmid cloning vectors replicating in Escherichia coli,amino acid-producing coryneform bacteria and Methylobacillus sp.

Summary Four hybrid plasmids were constructed from the cryptic plasmid pAM330 (from Brevibacterium lactofermentum; 4.5 kb) and the broadhost-range plasmid pGV1106 (9.0 kb; Km^r Sm^r) isolated from Escherichia coli. All of them were mobilized from E. coli into the Gram-negative methylotrophic bacterium Methylobacillus sp. and two of these constructs (pCEM300 and pCEM400) were transferred by transformation into B. flavum and Corynebacterium glutamicum. Their kanamycin-resistance determinant coming from Gram-negative hosts was expressed in these Gram-positive bacteria. Both pCEM300 and pCEM400 are very stably maintained in B. flavum and represent suitable vectors for gene cloning in coryneform producers of amino acids.     

Regulation of branched-chain amino acid biosynthesis in Streptomyces fradiae,a producer of tylosin

Synthesis of threonine dehydratase in Streptomyces fradiae was positively influenced by valine and negatively by isoleucine. However, these two amino acids had no effect on the activity of this enzyme. Synthesis of threonine dehydratase in -aminobutyrate resistant mutants of S. fradiae was pronouncedly less sensitive to the positive effect of valine and this change in regulation led to valine overproduction. Synthesis of acetohydroxy acid synthase is regulated in a similar manner to that of threonine dehydratase, however a lower level of expression was detected in -aminobutyrate resistant mutants. And again, no effect of branched-chain amino acids on acetohydroxy acid synthase activity was observed. It follows that in S. fradiae synthesis of threonine dehydratase is the main regulatory mechanism governing production and the mutual ratio of synthesized valine and isoleucine.Abbreviations -AB -aminobutyrate - AHAS acetohydroxy acid synthase - -KB -ketobutyrate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - TD threonine dehydratase - Trans. B. transaminase of branched-chain amino acids - VDH valine dehydrogenase     

Structural comparison of the 68 kDa laminin-binding protein and 5'-nucleotidase from chicken muscular sources: evidence against a gross structural similarity of both proteins

The 68 kDa laminin-binding protein purified from chicken skeletal muscle and the ectoenzyme 5'-nucleotidase from chicken gizzard are both able to interact with laminin. They were both shown to possess a nearly identical amino acid composition. The 79 kDa glycosylated form of 5'-nucleotidase can be transformed into an enzymatically active form by treatment with endoglycosidase F (Endo F). Deglycosylated (Endo F-treated) 5'-nucleotidase exhibits an apparent molecular mass of 68 kDa. Using immunological and finger-printing techniques, both proteins were analysed to determine their structural relatedness. The results obtained indicate that both proteins are not identical but may posses a few common peptides of yet unknown sequence and length.     

The heat generated by yeast cultures with a mixed metabolism in the transition between respiration and fermentation

The heat generated by both batch and continuous cultures of the yeast K. fragilis was studied using a modified Bench Scale Calorimeter. Batch cultures were used to measure the heat dissipation rates and the heat yields during fully aerobic and completely anaerobic growth, whereas continuous cultures enabled, in addition, a quantitative study of heat dissipation rates during growth on mixed metabolism. In this case, the extent of fermentation versus respiration could be specified and controlled by varying the degree of oxygen limitation. The heat dissipated per unit biomass formed was highest for fully respirative catabolism and fell continuously to a much lower value typical of anaerobic cultures as the catabolism was shifted increasingly to the fermentative mode. The heat generated per mole of oxygen taken up stayed quite close to the fully aerobic value of 506 kJ mol(-1) even when a sizable fraction of the substrate available to catabolism was fermented. If the fraction of respiration in the metabolism is lowered beyond a certain threshold, the ratio of the heat generation to oxygen consumption starts to increase dramatically and finally tends to infinity for fully anaerobic growth. All experimental results were quantitatively analyzed and explained on the basis of a simple model which formally describes the cultures in terms of two parallel "chemical" reactions. In simple cases such as the one presented here, the model enables calculation of the whole stoichiometry of the culture from a single measured heat yield.     

Frequent derepression of G6PD and HPRT on the marsupial inactive X chromosome associated with cell proliferation in vitro

X chromosome dosage compensation in Marsupials is like that in eutherian mammals except that the paternal X chromosome is always inactive, and silence of this chromosome is not well maintained. We previously showed that the unstable inactivation of the paternal G6PD allele is associated with the lack of DNA methylation in the 5' CpG cluster. Even though this CpG island is unmethylated, the paternal allele (marked by an enzyme variant) is at least partially and often severely repressed in most tissues of the opossum, so that factors other than methylation must inactivate the locus. Here we report that when cell cultures are established from these tissues, the silent G6PD locus is depressed. Although often complete, the extent of derepression differs among tissues and within different cell types in the same tissue, and is not accompanied by obvious changes in the pattern of chromosome replication. Studies of the HPRT locus in these cells show that the paternal HPRT allele also derepresses in cultured cells. These observations suggest that without DNA methylation to maintain the silence of the locus, tissue or cell-specific factors act to repress the silent locus, but are unable to maintain inactivity through cell division, or are lost as cells proliferate in culture.     

Cell attachment properties of collagen type VI and arg-gly-asp dependent binding to its α2(VI) and α3(VI) chains

Twelve of sixteen different cell types including fibroblasts and tumor cells were able to attach and spread on substrates of pepsin-solubilized or intact collagen VI, and on its triple helical domain. Attachment and spreading were independent of soluble mediator proteins (fibronectin, laminin) and collagen VI was distinct from collagens I, IV and V in the cells with which it interacted. Many of the same cells bound and spread on substrates prepared from unfolded α2(VI) and α3(VI) chains but not on the α1(VI) chain. The interactions with the chains were inhibited by low concentrations (10–100 μM) of synthetic RGDS and RGDT but not RGES peptides while the binding of cells to pepsin-solubilized collagen VI was more than 20-fold less sensitive to these peptides. The data incidate that cells have the ability to bind to collagen VI in a specific manner suggesting a similar function for collagen VI in situ.