ABSTRACT
The biosyntheses of oligosaccharides and glycoconjugates are conducted by glycosyltransferases. These extraordinarily diverse and widespread enzymes catalyze the formation of glycosidic bonds through the transfer of a monosaccharide from a donor molecule to an acceptor molecule, with the stereochemistry about the anomeric carbon being either inverted or retained. Human ABO(H) blood group A α-1,3-N-acetylgalactosaminyltransferase (GTA) generates the corresponding antigen by the transfer of N-acetylgalactosamine from UDP-GalNAc to the blood group H antigen. To understand better how specific active-site-residue protons and hydrogen-bonding patterns affect substrate recognition and catalysis, neutron diffraction studies were initiated at the Protein Crystallography Station (PCS) at Los Alamos Neutron Science Center (LANSCE). A large single crystal was subjected to H/D exchange prior to data collection and time-of-flight neutron diffraction data were collected to 2.5â Å resolution at the PCS to â¼85% overall completeness, with complementary X-ray diffraction data collected from a crystal from the same drop and extending to 1.85â Å resolution. Here, the first successful neutron data collection from a glycosyltransferase is reported.
Subject(s)
ABO Blood-Group System/chemistry , N-Acetylgalactosaminyltransferases/chemistry , Neutron Diffraction , Neutrons , Catalysis , Crystallography , Crystallography, X-Ray/methods , Humans , Hydrogen Bonding , Proteins , ProtonsABSTRACT
Based on the findings obtained a conclusion was reached that cryosurgery is a worth-while exercise in patients of child-bearing age as a method enabling the menstrual function to be preserved. Electrodestruction of endometrium is to be targeted to patients of perimenopausal age to achieve amenorrhea. The aim of the above policy of managing patients is to enhance efficacy of treatment of hyperplastic processes in endometrium.
Subject(s)
Cryosurgery , Electrosurgery , Endometrial Hyperplasia/surgery , Endometrium/surgery , Adult , Female , Follow-Up Studies , Humans , Middle Aged , Treatment OutcomeABSTRACT
The Fab fragment of the hybridoma antibody (YsT9.1) specific to Brucella abortus has been crystallized on earth using both Linbro plates and ground-based models of the flight hardware, as well as in microgravity on board the space shuttle Discovery and the space station Mir. Large-scale experiments using Linbro plates gave two different crystal morphologies, pyramidal and rhomboid, depending on conditions. The pyramidal crystals proved to scatter X-rays to higher resolution, and conditions within the ground-based flight hardware for both Discovery and Mir were adjusted to produce crystals with this morphology. The experiment on Discovery produced large crystals in each of ten chambers. The experiment on Mir produced crystals in only one of the five assigned chambers, despite the fact that the simultaneous ground-based experiment produced large crystals in every corresponding chamber. Data collection was attempted for crystals from both space and ground-based experiments. Higher resolution data was obtained from crystals grown on Discovery than from either Mir or ground-based crystals, even though the crystals obtained from Discovery were smaller and forced to grow over a much shorter period of time because of the shorter length of the shuttle mission.
ABSTRACT
X-ray study of chicken cytosolic aspartate aminotransferase revealed conformational changes in the protein of two kinds: (1) a shift of the small domain adjacent to substrate-binding area due to interaction of the protein with two carboxyl groups of substrate and (2) a change in inclination of the coenzyme plane due to replacement of C = N bond of the coenzyme with Lys-258 by C = N bond with a substrate. An asymmetry in subunit behaviour is observed in both cases: the domain is shifted in one subunit and the coenzyme is rotated in other. Substrate-binding properties of each subunit are strictly dependent on the protein conformation in substrate-binding area.
Subject(s)
Aspartate Aminotransferases/metabolism , Protein Conformation , Animals , Chickens , Crystallization , Cytosol/enzymology , Mitochondria/enzymology , Models, Molecular , X-Ray DiffractionABSTRACT
The paper describes a method for producing gram-scale quantities of beef pancreatic ribonuclease A (RNase A) (EC 3.1.4.22) from commercial, amorphous and crystalline RNases. The method involves enzyme purification by chromatography on SP-Sephadex G-25, rechromatography on Bio-Rax 70 followed by concentration, storage and desalting in optimal conditions. In their properties the resultant preparations of RNase A are not inferior to the best commercial samples of the enzyme.
Subject(s)
Pancreas/enzymology , Ribonucleases/isolation & purification , Animals , Cattle , CrystallizationABSTRACT
Aspartate transaminase (EC 2.6.1.1., Asp-transaminase) has been studied extensively, and much is now known about its physico-chemical, catalytic and other properties. X-ray studies that can provide a structural foundation for the events that occur during the transamination reaction are under way on three species of Asp-transaminase: the cytosolic enzyme from pig and chicken hearts, and the mitochondrial chicken heart enzyme. We describe here the interpretation of an electron density map of Asp-transaminase from chicken heart cytosol at 3.5 A.
Subject(s)
Aspartate Aminotransferases , Myocardium/enzymology , Animals , Binding Sites , Chickens , Cytosol/enzymology , Hydrogen Bonding , Protein Conformation , Structure-Activity Relationship , X-Ray DiffractionSubject(s)
Aspartate Aminotransferases , Animals , Chickens , Myocardium/enzymology , X-Ray DiffractionABSTRACT
We have studied the spectral properties of RNAase A containing a phosphopyridoxyl residue at the epsilon-NH2 group of Lys-7 or Lys-14. The overall conformations of the native and modified enzymes were shown to be rather similar. All three proteins have similar circular dichroism spectra within the 220-300-nm region, and similar thermal transition temperatures. All the changes in the RNAase A molecule modified are located in close proximity to the alkylated lysine residue. The phosphopyridoxyl group of (P-Pxy)-epsilon-Lys-41-RNAase A is situated directly at the enzyme active site and is 25% butied in the protein globule. The P-pyridoxyl group of (P-Pxy)-epsilon-Lys-7-RNAase A was shown to be located in the vicinity of the active site and to be more exposed to the solvent. In the pyridoxyl phosphate absorption band, optical activity is induced in both proteins. Study of the pH dependence of the changes occurring in the circular dichroism and absorption spectra has shown that in the modified proteins, the pyridoxyl phosphate chromophore is rather sensitive to the ionic state of the surrounding medium and serves as a "reporter" group when the relationship between structure and function of the RNAase A active site is being investigated.
Subject(s)
Pyridoxal Phosphate , Ribonucleases , Binding Sites , Circular Dichroism , Hydrogen-Ion Concentration , Lysine , Protein Binding , Protein Conformation , Protein Denaturation , Spectrophotometry, Ultraviolet , TemperatureABSTRACT
The physico-chemical properties have been studied of RNase A selectively modified at the E-NH2-group of Lys-7 and Lys-41 with pyridoxal-P. Modification did not affect conformational stability of the protein globule, thus all changes in the molecule of the modified RNase A were localised around the alkylated Lys residue. In the both cases pyridoxyl-P. The residue was shown to be localized in the active site region of the (P-Pxy)-Lys-7-RNase A and its chromophore parts was highly exposed to the solvent. (P-Pxy) E-Lys-7-RNase A and its chromophore parts was highly exposed to the solvent. In the Lys-41 derivative, pyridoxamine-P was situated exactly in the active site and is partially hidden in the protein grobule. The pH-dependence of absorption spectra indicates that the chromophore of pyridoxyl-P in modified proteins is quite sensible to the ionic state of its surrounding. The usefulness of pyridoxyl-P as a reporter group was proved in the study with (P-Pxy)-Lys-7-RNase A. Some conformational changes involving His-119 were shown to take place in the course of the enzyme-nucleotide complex formation.
