Conformational states of (K+ + H+)-ATPase studied using tryptic digestion as a tool

The (K+ + H+)-ATPase from gastric mucosa has been treated by limited proteolytic digestion with trypsin to study the conformational states of the enzyme. The existence of a K+- and an ATP-form of the enzyme follows from the kinetics of inactivation and from the specific cleavage products. In the presence of K+ the 95 kDa chain is cleaved into two fragments of 56 and 42 kDa, whereas in the presence of ATP fragments of 67 and 35 kDa are formed. When Mg2+ is present during tryptic digestion cleavage products which are specific for both the ATP- and the K+-form of the enzyme are yielded. In analogy to ATP, Mg2+ is able to convert the enzyme from a K+-conformation to a more protected form. Moreover Mg2+ supports the protecting effect of ATP against tryptic inactivation. The K0.5 for ATP is lowered from 1.6 mM (no Mg2+) to 0.2 mM in the presence of 10 mM Mg2+. Mg2+, which in previous studies has been shown to induce a specific conformation, apparently induces a conformation different from the K+-form of the enzyme and has ATP-like effects on the enzyme. In addition it has been found that in the initial rapid phase of the digestion process the K+-ATPase activity is interrupted at a step which is very likely the interconversion of the phosphoenzyme forms E1P and E2P, since neither the K+-stimulated p-nitrophenylphosphatase activity nor the phosphorylation of the enzyme are inhibited in this phase. During the tryptic digestion in the presence of K+ there is a good correlation between the residual ATPase activity and the amount of the catalytic subunit left, suggesting that the latter is homogeneous. After tryptic digestion in the presence of K+, phosphorylation only occurs in the 42 kDa and not in the 56 kDa band. The same experiments in the presence of ATP yield only phosphorylation in the 67 kDa band and not in the 35 kDa band. A provisional model for the structure of the catalytic subunit is given.     

Limiting factors for seed production in Cynoglossum officinale

Summary Over three years of study, small plants of Cynoglossum officinale consistently produced more flowers per unit of dry weight than large plants. In contrast to earlier results, weight of all seeds tended to increase more than proportional to size. As a result a positive correlation existed between seed set per flower and plant size. The correlation between the mean number of pollinator visits per flower and size was positive but not significant. In a field experiment we found that resources rather than pollen were limiting seed set. Thus, it is unlikely that enhanced pollination of the largest plants causes the size-dependency of seed set per flower. Alternative hypotheses are discussed briefly.Publication of the Meijendel Comité, New Series No. 96     

Linkage analysis of chromosome 17 markers in British and South African families with neurofibromatosis type 1

Nine markers from the pericentromeric region of chromosome 17 were typed in 16 British and five South African families with neurofibromatosis type 1 (NF1). The markers--p17H8, pHHH202, and EW204--were linked to NF1 at recombination fractions less than 1%. No evidence of locus heterogeneity was detected. Inspection of recombinant events in families informative for several markers suggests that the NF1 gene is located between the markers EW301 (cen-p11.2) and EW206 (cen-q12) and possibly distal to pHHH202 (q11.2-q12).     

Effects of oxygen on the growth and metabolism of Actinomyces viscosus

Abstract Actinomyces viscosus is a predominant microorganism in dental plaque. It is, just as the oral Streptococcus spp., a saccharolytic and aero-tolerant organism. We have investigated the effects of oxygen on the growth and metabolism of A. viscosus . To this end A. viscosus Ut 2 was grown in a glucose limited chemostat culture on a chemically defined medium ( D = 0.2 h⁻¹) with exposure to variable amounts of oxygen. The Y_glucose increased from 62.5 g · mol⁻¹ under anaerobic conditions to 149 g · mol⁻¹ under aerobic conditions, while, concomitantly, the carbon recovery from acidic fermentation products decreased from 75% to 7%. Addition of [¹⁴C]glucose to the chemostat showed that the glucose, which was not converted to acidic fermentation products, was instead converted to carbon dioxide or used for the production of biomass. Under aerobic and anaerobic conditions identical cytochrome spectra, containing only two cytochrome b -type absorption bands, were found. It was concluded that electron transport phosphorylation probably occurs both under aerobic and anaerobic conditions. Anaerobically, fumarate served as the electron acceptor, while the high growth yields observed under aerobic conditions are likely to be explained by citric acid cycle activity coupled to electron transport phosphorylation.     

Signal peptidase I of Bacillus subtilis: patterns of conserved amino acids in prokaryotic and eukaryotic type I signal peptidases.

Signal peptidases (SPases) remove signal peptides from secretory proteins. The sipS (signal peptidase of subtilis) gene, which encodes an SPase of Bacillus subtilis, was cloned in Escherichia coli and was also found to be active in E.coli. Its overproduction in B.subtilis resulted in increased rates of processing of a hybrid beta-lactamase precursor. The SipS protein consisted of 184 amino acids (mol. wt 21 kDa). The protein showed sequence similarity with the leader peptidases of E.coli and Salmonella typhimurium, and the mitochondrial inner membrane protease I of Saccharomyces cerevisiae. Patterns of conserved amino acids present in these four proteins were also detected in the Sec11 subunit of the SPase complex of S.cerevisiae and the 18 and 21 kDa subunits of the canine SPase complex. Knowledge of the sequence of SipS was essential for the detection of these similarities between prokaryotic and eukaryotic SPases. The data suggest that these proteins, which have analogous functions, belong to one class of enzymes, the type I SPases.     

Induction of interdigitating reticulum cell-like differentiation in human monocytic leukemia cells by conditioned medium from IL-2-stimulated helper T-cells

Monocytic leukemia (MoL) cells were obtained from the peripheral blood of a patient in whom the leukemic cells infiltrating various lymphoreticular organs exhibited features intermediate between interdigitating reticulum cells (IDC) and ordinary phagocytic macrophages, whereas the leukemic cells in the peripheral blood were essentially monocytic and lacked such features. Peripheral blood CD4+ T-cells were established as an interleukin-2-dependent T-cell line. When the MoL cells were exposed for a few days to conditioned medium from the T-cell line, they extended several dendritic cytoplasmic projections and became intensely positive for HLA-DR antigen, cytoplasmic S-100β protein, and CD1 antigen. Functionally, the conditioned medium significantly down-regulated Fc-mediated and Fc-independent phagocytic activities, and the levels of lysosomal enzymes such as lysozyme and nonspecific esterase in the MoL cells. Moreover, the conditioned medium significantly up-regulated the accessory cell function of the MoL cells as measured by the primary allogenic mixed leukocyte reaction (MLR). Furthermore, the conditioned medium significantly down-regulated the expression of CD14 antigen. Biochemical analysis indicated that the factor responsible for these changes is a protein which is distinct from known human cytokines and whose molecular weight is approximately 31 kDa. These findings suggest that IDC are closely related the monocytic lineage and that helper T-cells play an important role in constructing the microenvironment of T-lymphoid tissues which is necessary for the differentiation and maturation of IDC.     

Signal peptidase I overproduction results in increased efficiencies of export and maturation of hybrid secretory proteins in Escherichia coli

Summary The effects of 25-fold overproduction ofEscherichia coli signal peptidase I (SPase I) on the processing kinetics of various (hybrid) secretory proteins, comprising fusions between signal sequence functions selected from theBacillus subtilis chromosome and the mature part of TEM-β-lactamase, were studied inE. coli. One precursor (pre[A2d]-β-lactamase) showed an enhanced processing rate, and consequently, a highly improved release of the mature enzyme into the periplasm. A minor fraction of a second hybrid precursor (pre[Al3i]-β-lactamase), which was not processed under standard conditions of SPase I synthesis, was shown to be processed under conditions of SPase I overproduction. However, this did not result in efficient release of the mature β-lactamase into the periplasm. In contrast, the processing rates of wild-type pre-β-lactamase and pre(A2)-β-lactamase, already high under standard conditions, were not detectably altered by SPase I overproduction. These results demonstrate that the availability of SPase I can be a limiting factor in protein export inE. coli, in particular with respect to (hybrid) precursor proteins showing low (SPase I) processing efficiencies.     

Application of good laboratory practice (GLP) to culture collections of microbial and cell cultures

Although the principles and the necessity for good laboratory practice (GLP) guidelines to confirm the credibility, integrity, and quality of non-clinical laboratory studies have been known for more than a decade, culture collection activities are not subject to them. Because of recent advances in biotechnology, culture collections face increased demands not only for quality cultures but also current information. When applied in culture collections, GLP guidelines prove to be an excellent management tool as well as a cost-effective system of providing authentic and reliable microbial and cell cultures and associated data.