Crystallization of beta-galactosidase from Escherichia coli.

Two crystal forms of beta-galactosidase have been obtained from Escherichia coli. One crystal form is hexagonal space group P6222 or enantiomorph, with cell dimensions a = b = 154 A, c = 750 A. The second form is monoclinic, space group P21, with cell dimensions a = 107.9 A, b = 207.5 A, c = 509.9 A, beta = 94.7 degrees. The monoclinic form seems better suited to detailed structural analysis. The crystals are radiation-sensitive, but by using synchrotron radiation in conjunction with a long (400 mm) crystal-to-film distance it was possible to resolve the individual reflections. On the basis of crystal density measurements, there are four tetramers each of molecular weight 465,000 per asymmetric unit. The Patterson function strongly suggests that two of the tetramers are related to the other two by translation. The data are consistent with the tetramers having 222 point symmetry, but this is not proven.     

beta-Furfuryl-beta-Glucoside: An Endogenous Activator of Higher Plant UDP-Glucose:(1-3)-beta-Glucan Synthase : Biological Activity, Distribution, and in Vitro Synthesis

In a recent paper (P Ohana, DP Delmer, JC Steffens, DE Matthews, R Mayer, M Benziman [1991] J Biol Chem 266: 13472-13475), we described the purification and structural characterization of β-furfuryl-β-glucoside (FG), an endogenous activator of plant UDP-glucose:(1→3)-β-glucan (callose) synthase. In the present report, we provide evidence that FG specifically stimulates callose synthase. The effects of FG on the kinetic properties of callose synthase were studied, and we ascertained that FG, or at least a very similar compound, is present in other plant systems. Chemically synthesized α-furfuryl-β-glucoside also stimulates callose synthase, exhibiting a slightly higher K_a of 80 micromolar, compared with 50 micromolar for FG. In addition, we have identified and partially characterized an enzyme that catalyzes the synthesis of FG using β-furfuryl alcohol and UDP-glucose as substrates. A model for the regulation of callose synthesis in vivo, involving changes in intracellular compartmentation of FG and Ca²⁺, is proposed.     

Fetuin: an acute phase protein in cattle

Summary Fetuin is a plasma protein present in high concentrations during fetal development in animals of the order Artiodactyla. Its role is not known. The human homologue of fetuin — ₂HS glycoprotein — has been shown to be a negative acute phase protein in adult plasma. In the present study, the concentration of fetuin was measured in the serum of healthy cattle (Bovis bovis) and in animals with various injuries and inflammatory disorders. The levels were decreased by 30% in pregnancy but increased up to 10-fold in some trauma cases. A significant negative correlation between the concentrations of fetuin and albumin has also been found. Thus, fetuin appears to be a positive acute phase protein in cattle.Abbreviations ₂HS ₂HS glycoprotein - AP acute phase     

Two new photoaffinity polyamines appear to alter the helical twist of DNA in nucleosome core particles

Two new photoaffinity derivatives of polyamines have been synthesized by the reaction of spermine or spermidine with methyl 4-azidobenzimidate. The new compounds were purified chromatographically and characterized by several methods including proton magnetic resonance spectroscopy. The spermine derivative is N1-ABA-spermine [(azidobenzamidino)spermine], and the spermidine derivative is a mixture of N1- and N8-ABA-spermidine. ABA-spermine stabilizes nucleosome core particles in thermal denaturation experiments, with similar but not identical effects when compared with the parent polyamine, spermine. In circular dichroism experiments, ABA-spermine was capable of producing a B----Z transition in poly(dG-m5dC) at a concentration of 30 microM, compared with 5 microM required to produce the same effect with spermine. On the other hand, ANB-spermine [(azidonitrobenzoyl)spermine; Morgan, J. E., Calkins, C. C., & Matthews, H. R. (1989) Biochemistry 28, 5095-5106] stabilized the B form of poly(dG-br5dC). ABA-spermine is a potent inhibitor of ornithine decarboxylase from Escherichia coli, giving 50% inhibition at 0.12 mM, while ANB-spermine is a modest inhibitor, comparable to spermine or spermidine. Under conditions of nitrogen-limited growth, yeast take up ABA-spermine and ABA-spermidine at approximately one-third to half the rate of spermidine or spermine. In contrast, ANB-spermine was not significantly taken up. The photoaffinity polyamines were used to photoaffinity label the DNA in nucleosome core particles, and the sites of labeling were determined by exonuclease protection. All photoaffinity reagents showed both nonspecific labeling and specific sites of higher occupancy. However, the positions of the sites varied: the ANB-spermine sites confirmed those previously reported (Morgan et al., 1989); the ABA-spermine and ABA-spermidine sites were spaced at 9.8 bp intervals from the 3' end of each DNA strand. This observation, together with the effect of spermine on the circular dichroism of DNA in nucleosome core particles, implies that polyamines alter the helical twist of DNA in nucleosome core particles. The ABA-polyamines are offered as general-purpose photoaffinity polyamine reagents.     

Crystallographic determination of the mode of binding of oligosaccharides to T4 bacteriophage lysozyme: Implications for the mechanism of catalysis

Phage lysozyme has catalytic activity similar to that of hen egg white lysozyme, but the amino acid sequences of the two enzymes are completely different.The binding to phage lysozyme of several saccharides including N-acetylglucosamine (GlcNAc), N-acetylmuramic acid (MurNAc) and (GlcNAc)₃ have been determined crystallographically and shown to occupy the pronounced active site cleft. GlcNAc binds at a single location analogous to the C site of hen egg white lysozyme. MurNAc binds at the same site. (GlcNAc)₃ clearly occupies sites B and C, but the binding in site A is ill-defined.Model building suggests that, with the enzyme in the conformation seen in the crystal structure, a saccharide in the normal chair configuration cannot be placed in site D without incurring unacceptable steric interference between sugar and protein. However, as with hen egg white lysozyme, the bad contacts can be avoided by assuming the saccharide to be in the sofa conformation. Also Asp20 in T4 lysozyme is located 3 Å from carbon C₍₁₎ of saccharide D, and is in a position to stabilize the developing positive charge on a carbonium ion intermediate. Prior genetic evidence had indicated that Asp20 is critically important for catalysis. This suggests that in phage lysozyme catalysis is promoted by a combination of steric and electronic effects, acting in concert, The enzyme shape favors the binding in site D of a saccharide with the geometry of the transition state, while Asp20 stabilizes the positive charge on the oxocarbonium ion of this intermediate. Tn phage lysozyme, the identity of the proton donor is uncertain. In contrast to hen egg white lysozyme, where Glu35 is 3 Å from the glycosidic DOE bond, and is in a non-polar environment, phage lysozyme has an ion pair, Glull … Arg145, 5 Å away from the glycosidic oxygen. Possibly Glull undergoes a conformational adjustment in the presence of bound substrate, and acts as the proton donor. Alternatively, the proton might come from a bound water molecule.     

Aminoacyl-transfer RNA populations in mammalian cells chromatographic profiles and patterns of codon recognition.

The chromatographic profiles of 20 aminoacyl-tRNAs from rabbit liver were compared to those of rabbit reticulocytes by reverse phase chromatography and the chromatographic profiles of 20-aminoacyl-tRNAs from bovine liver were compared to those of bovine brain. The two rabbit tissues showed significant differences in the elution profiles of most aminoacyl-tRNAs, while the elution profiles of the aminoacyl-tRNAs from the bovine tissues showed fewer differences. The patterns of codon recognition of several aminoacyl-tRNAs fractionated from rabbit reticulocytes have also been compared to those fractionated from rabbit liver.