Integral kinetics of alpha-galactosidase purified from Glycine max for simultaneous hydrolysis of stachyose and raffinose

alpha-Galactosidase from soybean (Glycine max) was purified by a five-step procedure. The enzyme's natural substrates, raffinose and stachyose, have K(m)'s of 3. 0 mM and 4. 79 mM, respectively. The products, galactose and sucrose, were measured after separation by liquid chromatography. Galactose is a competitive product inhibitor of stachyose and raffinose hydrolysis with a K(i) of 0. 12 mM. We determined these parameters by an integral kinetic approach. Stachyose hydrolysis gives a nearly constant level of raffinose shortly after hydrolysis begins. Thus, cleavage of the first alpha-(1,6)-bond in the tetrasaccharide is the rate-limiting step. Since the stachyose hydrolysis yields raffinose, soybean alpha-galactosidase simultaneously hydrolyzes two substrates. We present a novel approach for analyzing simultaneous substrate hydrolysis with competitive product inhibition by a modified integral rate expression. The experimentally found kinetic parameters are confirmed by solving the simultaneous equations which describe the hydrolysis. This technique may be applicable to other hydrolytic enzymes with multiple substrates.     

Muscle provocation test. A sensitive method for discrimination between carriers and noncarriers of Duchenne muscular dystrophy

Summary After 40 min of strenuous exercise on a bicycle ergometer (muscle provocation test, MPT), normal women and obligate carriers showed a progressive elevation of serum creatine kinase, with a peak 8 h after exercise. The diagnostic applicability of MPT for carrier detection is demonstrated.     

Sequence homology between the tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Escherichia coli and hemerythrin from Sipunculida

The first enzyme of the common aromatic biosynthetic pathway in Escherichia coli, the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, contains iron as an integral part of the polypeptide chain, and the enzyme shows an absorption maximum around 350 nm (McCandliss, R.J., and Herrmann, K.M. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 4810-4813). These two properties are also found in hemerythrin, the oxygen carrier of certain marine invertebrates. The amino acid sequence of residues 10 to 18 of the enzyme from E. coli, His-Ile-Thr-Asp-Glu-Gln-Val-Leu-Met, is highly homologous to the sequence of residues 54 to 62 of hemerythrin from Phascolopsis gouldii, His-Phe-Leu-Asn-Glu-Gln-Val-Leu-Met. His54 and Glu58 of hemerythrin have previously been identified through x-ray and protein sequence analysis as iron ligands. We suggest that residues 10 to 18 of the E. coli enzyme represent part of the iron binding fold in this protein, and that His10 and Glu14 are iron ligands.     

Larvalentwicklung und Metamorphose vonPhoronis psammophila (Phoronida,Tentaculata)

The larval development ofPhoronis psammophila Cori is divided into 6 phases (on the basis of increasing pairs of larval tentacles); furthermore an initial and a ripe phase are distinguished. Specific aspects of the development are described: Formation and structure of larval tentacles; anlage of adult tentacles as a thickening in the larval tentacle base; late development of the metasome (larva with 4–6 tentacles); formation of the metasome pouch in the larva with 8 tentacles; enlargement of the apical plate; differentiation of the gut; differentiation of larval nephridia; formation of pigment particles in the larva with 6 tentacles (storage function of pigments and its significance for larval identification); different types of discoflagella in various regions of the body. The larval development shows the following tendencies: Improvement of locomotion; intensification of food filtration; anlage of adult organs in the larva leading to a shortening of metamorphosis duration. The larva ofP. psammophila is compared with those ofP. pallida, P. hippocrepia, andP. vancouverensis. Earlier larval determinations ofP. psammophila (e.g.Actinotrocha sabatieri, A. hatschekii) are shown to have been mistakes. Termination of the postembryonic phase (metamorphosis) can be induced experimentally by bacteria and also by cations. Pure or mixed bacteria cultures must be present at the beginning exponential growth phase. The bacteria density required is 20–94×10⁶ bact.ml^?1 for pure cultures and on the average 28×10⁶ bact. ml^?1 for mixed cultures. Metamorphosis initiation by cations can be induced with CsCl (0.06 M) and RbCl (0.035 M). Metamorphosis ofP. psammophila occurs in 6 phases: larva, ready for metamorphosis; larva, activated by bacteria or ions; evagination of the metasome diverticle, dislocation of gut; losing and swallowing of episphaere and larval tentacles; formation of the youngP. psammophila. All developmental phases are described and compared with those ofP. muelleri; imperfect metamorphosis is characterized and the youngP. psammophila compared with older stages and the adult Phoronis.     

Yeast dipeptidase: active site mapping by kinetic studies with substrates and substrate analogs.

In order to characterize the active site of yeast dipeptidase in more detail, kinetic studies with a variety of dipeptide substrates and substrate analogs were performed. To analyze kinetic data, computer programs were developed which first calculate initial velocities from progress curves and then evaluate the kinetic parameters by nonlinear regression analysis. A free carboxyl group is a prerequisite for binding of dipeptidase substrates; its position relative to the peptide bond must not deviate from the normal L-dipeptide conformation. The spatial arrangement of the terminal ammonium ion seems to be less crucial. The enzyme's substrate specificity clearly reflects the interactions of the substrate amino acid side chains with complementary dipeptidase subsites. The domain of the enzyme in contact with the C-terminal substrate side chain seems to be an open structure of moderately hydrophobic character. In contrast, the binding site for the amino-terminal side chain is a more strongly hydrophobic "pocket" of limited dimensions. The kinetics of inhibition by free amino acids points to an ordered release of products from the enzyme.     

Cis-regulatory characterization of sequence conservation surrounding the Hox4 genes

Hox genes are key regulators of anterior-posterior axis patterning and have a major role in hindbrain development. The zebrafish Hox4 paralogs have strong overlapping activities in hindbrain rhombomeres 7 and 8, in the spinal cord and in the pharyngeal arches. With the aim to predict enhancers that act on the hoxa4a, hoxb4a, hoxc4a and hoxd4a genes, we used sequence conservation around the Hox4 genes to analyze all fish:human conserved non-coding sequences by reporter assays in stable zebrafish transgenesis. Thirty-four elements were functionally tested in GFP reporter gene constructs and more than 100 F1 lines were analyzed to establish a correlation between sequence conservation and cis-regulatory function, constituting a catalog of Hox4 CNEs. Sixteen tissue-specific enhancers could be identified. Multiple alignments of the CNEs revealed paralogous cis-regulatory sequences, however, the CNE sequence similarities were found not to correlate with tissue specificity. To identify ancestral enhancers that direct Hox4 gene activity, genome sequence alignments of mammals, teleosts, horn shark and the cephalochordate amphioxus, which is the most basal extant chordate possessing a single prototypical Hox cluster, were performed. Three elements were identified and two of them exhibited regulatory activity in transgenic zebrafish, however revealing no specificity. Our data show that the approach to identify cis-regulatory sequences by genome sequence alignments and subsequent testing in zebrafish transgenesis can be used to define enhancers within the Hox clusters and that these have significantly diverged in their function during evolution.