Novel kinetic and structural properties of the class-I D-fructose 1,6-bisphosphate aldolase from Escherichia coli (Crookes'' strain).

Investigation of aldolase 1, the class-I D-fructose 1,6-bisphosphate aldolase (EC4.1.2.13) from Escherichia coli (Crookes' strain), showed it to have unusual kinetic and structural properties. The enzyme appeared to be larger than was previously supposed and may be a decamer with a mol. wt. of approx. 340000. Its fructose 1,6-bisphosphate-cleavage activity was unaffected by these compounds. The enhancement exhibited a strong dependence on pH. These novel kinetic properties do not seem to be shared by any other fructose 1,6-bisphosphate aldolase, but recall the activation by polycarboxylic acids of the deoxyribose 3-phosphate aldolases from some other organisms. In view of its unusual properties, it is unlikely that aldolase 1 from E. coli is closely related to the class-1 aldolases that have been detected in several other prokaryotes, or to the typical class-1 enzymes from eukaryotes.     

Effects of Combinations of Substrates on Maximum Growth Rates of Several Rumen Bacteria

Five rumen bacteria, Selenomonas ruminantium, Bacteroides ruminicola, Megasphaera elsdenii, Butyrivibrio fibrisolvens, and Streptococcus bovis were grown in media containing nonlimiting concentrations of glucose, sucrose, maltose, cellobiose, xylose and/or lactate. Each bacterium was grown with every substrate that it could ferment in every possible two-way combination. Only once did a combination of substrates result in a higher maximum growth rate than that observed with either substrate alone. Such stimulations of growth rate would be expected if specific factors unique to individual substrates (transport proteins and/or enzymes) were limiting. Since such synergisms were rare, it was concluded that more general factors limit maximum growth rates in these five bacteria.     

A spectroscopic study of metal ion and ligand binding to β-lactamase II

β-Lactamase II has two metal-binding sites. The electronic spectra of Cd(II)- and Co(II)-substituted β-lactamase II have been investigated. It is suggested that a thiol ligand is involved in metal binding at the first site. The stoichiometric dissociation constants for Co(II) binding to β-lactamase II were estimated to be 0.13 and 2.66 mM (pH 6.0, 4°C, 1 M NaCl) by equilibrium dialysis. Competition between Zn(II) and Co(II) for the first metal binding site suggests a value of 0.7 μM (pH 6.0, 30°C, 1 M NaCl) for the dissociation constant o Zn(II).The electronic spectra of the Co(II) enzyme lead to the suggestion that the coordination geometries around the metal ions in the first and second sites are related to those of a distorted tetrahedron and octahedron, respectively.     

The rate of interconversion between the two unfolded forms of ribonuclease A does not depend on guanidinium chloride concentration.

The interconversion between the fast-folding and slow-folding forms of ribonuclease A is unaffected by the protein denaturant guanidinium chloride, between 2.8 m and 7.0 m, at 10 °C. Thus the rate of this reaction is insensitive to denaturants, in contrast to the model proposed by Kanehisa &; Tsong (1979). This result is consistent with other evidence that the interconversion reaction is proline isomerization.     

Nuclear magnetic resonance evidence for a structural intermediate at an early stage in the refolding of ribonuclease A

The kinetics of refolding of heat-unfolded ribonuclease A have been studied by Fourier transform proton nuclear magnetic resonance at 10 °C, pH 2. A single refolding reaction is observed: it corresponds to the slow-refolding reaction seen in stopped-flow studies of refolding at higher temperatures. There are two results of interest for the mechanism of protein folding. (1) A new resonance (X) is observed that shows the presence of a structural intermediate in refolding. (2) The α-helix close to the N-terminal end of ribonuclease A apparently forms rapidly when the unfolded protein is brought to refolding conditions.The folding intermediate has been studied by monitoring the C-2 protons of the four histidine residues. The intermediate contains one residue (X) in a partly folded environment and the other three residues in unfolded environments. The composite resonance (U) of these three protons at 10 °C agrees with the average chemical shift of the histidine residues in heat-unfolded ribonuclease A at high temperatures. During refolding at 10 °C, the resonance intensities of U and X disappear at the same rate that the spectrum of native ribonuclease A is regained.Partial deuteration experiments show that X is either histidine 12 or 119. Comparative studies of the amino-terminal fragment 1–20 of ribonuclease A indicate that X is histidine 12. The appearance of structure in this peptide can be followed by temperature-dependent changes in the chemical shift of histidine 12. At 10 °C the chemical shifts of histidine 12 and X agree closely. These results are consistent with the circular dichroism study of peptide 1–13 by Brown &; Klec (1971), who concluded that helix formation occurs at low temperatures.     

Circulating immune complexes in patients with breast cancer.

In a retrospective study in women with breast cancer circulating immune complex levels were measured by radioimmunoprecipitation with 125I-Clq. Before operation all the patients showed plasma immune complex levels significantly higher than those in controls. Twelve months after mastectomy patients identified clinicopathologically as having a good prognosis had almost normal levels of immune complexes. By contrast, patients with detectable dissemination on diagnosis or those who died within 22 months after mastectomy had significantly raised plasma levels. The tumour-specific nature of the immune complexes detected remains to be shown and suggestions about the applicability of this test not only for prognosis but also for monitoring the course of malignant diseases need to be confirmed by further investigations.     

Phosphorylation of a synthetic gastrin peptide by the tyrosine kinase of A431 cell membranes

The peptide Arg.Arg.Leu.Glu.Glu.Glu.Glu.Glu.Ala.Tyr.Gly was synthesized as an analogue of residues 20–30 of human gastrin 34. The epidermal growth factor-stimulated tyrosine kinase of A431 cell membranes phosphorylated the peptide's single tyrosine residue. K_m values of 0.11 and 0.61mM and V_max values of 1.71 and 0.68nmol/min/mg were obtained in the presence and absence of epidermal growth factor respectively. This is the first report of phosphorylation of tyrosine in a sequence related to a human hormone.