Multicentric reticulohistiocytosis.

A 41 -year-old man presented with multiple, painful and tender nodules on the palms, soles, scalp; and on the limbs predominantly around the joints with associated arthropathies. Smaller nodules were seen on the ear helices. There was no other clinically evident or investigative abnormality. Histopathological study confirmed a diagnosis of multicentric reticulo histiocytosis.

Affinity properties of phosvitin: interaction of phosvitin with serine hydroxymethyl transferase.

The affinity of phosvitin with serine hydroxymethyl transferase (SHMT), an acidic multi-subunit protein, was evaluated by measurements of enzyme activity, sedimentation velocity, steady-state fluorescence, circular dichroism and kinetic thermal stability. While the presence of phosvitin had no effect on the SHMT activity, the sedimentation coefficient of SHMT increased from 8.7 S to 12.5 S suggesting the formation of a complex at a SHMT:phosvitin molar ratio of 2:1. Based on steady-state fluorescence quenching measurements an association constant of 2.4 +/- 0.2 x 10(5) M-1 at 25 degrees C was obtained for the interaction of phosvitin with SHMT. The temperature dependency of the association constant in the range 15-35 degrees C suggests the involvement of ionic forces in the interaction. The thermal inactivation of SHMT followed first order kinetics. In the presence of phosvitin the rate constant decreased and half time increased. The circular dichroism measurements suggest that phosvitin interaction does not involve pyridoxal phosphate binding domain of the enzyme. Although minor changes in the secondary structure of the enzyme were observed, the environment around aromatic amino acids did not change significantly.

A stoichiometric analysis of bovine serum albumin-gossypol interactions: a fluorescence quenching study.

The interaction of gossypol with bovine serum albumin, human serum albumin and n-bromosuccinimide-modified bovine serum albumin has been followed by fluorescence quenching measurements. The presence of a high affinity site (association constant K = 2.2 x 10(6) M-1) for gossypol on bovine serum albumin and human serum albumin is indicated. The stoichiometry of binding for the high affinity site was evaluated using Job's method of continuous variation, thereby suggesting the formation of 1:1 complex. Modification of the tryptophan residues on bovine serum albumin does not affect the binding of gossypol to either high or low affinity site of albumin.

Interactions of methoxyamine with pyridoxal-5'-phosphate-Schiff's base at the active site of sheep liver serine hydroxymethyltransferase.

The mechanism of interaction of methoxyamine with sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) (SHMT) was established by measuring changes in enzyme activity, visible absorption spectra, circular dichroism and fluorescence, and by evaluating the rate constant by stopped-flow spectrophotometry. Methoxyamine can be considered as the smallest substituted aminooxy derivative of hydroxylamine. It was a reversible noncompetitive inhibitor (Ki = 25 microM) of SHMT similar to O-amino-D-serine. Like in the interaction of O-amino-D-serine and aminooxyacetic acid, the first step in the reaction was very fast. This was evident by the rapid disappearance of the enzyme-Schiff base absorbance at 425 nm with a rate constant of 1.3 x 10(3) M-1 sec-1 and CD intensity at 430 nm. Concomitantly, there was an increase in absorbance at 388 nm (intermediate I). The next step in the reaction was the unimolecular conversion (1.1 x 10(-3) sec-1) of this intermediate to the final oxime absorbing at 325 nm. The identity of the oxime was established by its characteristic fluorescence emission at 460 nm when excited at 360 nm and by high performance liquid chromatography. These results highlight the specificity in interactions of aminooxy compounds with sheep liver serine hydroxymethyltransferase and that the carboxyl group of the inhibitors enhances the rate of the initial interaction with the enzyme.

Effect of chemical modification on struture and activity of glucoamylase from Aspergillus candidus and Rhizopus species.

The histidine, tyrosine, tryptophan and carboxyl groups in the enzyme glucoamylase from Aspergillus Candidus and Rhizopus species were modified using group specific reagents. Treatment of the enzyme with diethylpyrocarbonate resulted in the modification of 0·3 and 1 histidine residues with only a slight loss in activity (10% and 35%) of glucoamylase from Aspergillus candidus and Rhizopus species respectively. Modification of tyrosine either by Nacetylimidazole or [I125]-leads to a partial loss of activity. Under denaturing conditions, maltose did not help in protecting the enzyme against tyrosine modification or inactivation. Treatment with 2-Hydroxy-5-nitro benzyl bromide in the presence of urea, photooxidation at pH 9·0, N-bromosuccinamide at pH 4·8 resulted in a complete loss of activity· However, the results of experiments in the presence of maltose and at pH 4·8 photooxidation and Nbromosuccinamide treatment suggested the presence of two tryptophan residues at the active site. There was a complete loss of enzyme activity when 10 and 28 carboxyl groups from Aspergillus candidus and Rhizopus, respectively were modified. Modification in the presence of substrate maltose, showed at least two carboxyl groups were present at the active site of enzyme and that only one active center seems to be involved in breaking ally 3 types of α-glucosidic linkages namely α-1,4, α-1, 6 and α-l,3.

Fungal glucoamylases.

The purification and properties of glucoamylase (α-l,4-glucan glucohydrolase, EC 3.2.1.3) from different fungal sources have been compared. The studies on the conformation and activity of the native enzyme at a function of pH, temperature, substrate concentration and the effect of denaturants and on the role of carbohydrate moiety on structure and stability have been reviewed. The chemical modification of the active centre, binding kinetics of the substrate and active site and the mechanism of action have been summarized. They differ in their fine structure as revealed by their near ultra-violet circular dichroism spectra and contain 30–35 % α-helix, 24–36 % β-structure and the rest aperiodic structure. The activity of the enzyme is very sensitive to the environment around aromatic aminoacid residues. The glucoamylases are glycoprotein in nature, differ in their content and nature of carbohydrate from different sources. The carbohydrate moiety plays an important role in stabilising the native conformation of the enzyme and is not involved in activity and antigenecity. At the active site of the enzyme, two tryptophan and two carboxyl (glutamate or aspartate) groups are present. It is likely that the histidine and tyrosine residues which are present away from the active site are involved in binding of the substrate. There seems to be seven subsites which are involved in binding of the substrate and the catalytic site is situated in between 1 and 2 subsites. In breaking of α-1,4-, α-1,3-, and α-l,6-bonds only one active centre is involved. Studies on the immobilization of either glucoamylase alone or as a part of a multienzyme system have been reviewed briefly.

Structure and stability of glucoamylase II from Aspergillus niger: A circular dichroism study.

Glucoamylase II (EC 3.2.1.3) from Aspergillus niger has 31 % α-helix, 36 % ßstructure and rest aperiodic structure at pH 4·8 as analysed by the method of Provencher and Glockner (1981, Biochemistry, 20,33). In the near ultra-violet circular dichroism spectrum the enzyme exhibits peaks at 304, 289, 282 and 257 nm and troughs at 285, 277 and 265 nm respectively. The enzyme activity and structure showed greater stability at pH 4·8 than at pH 7·0, were highly sensitive to alkaline pH but less sensitive to acid pH values. The enzyme retained most of its catalytic activity and structure even on partial removal of carbohydrate moieties by periodate treatment but was less stable at higher temperatures and storage at 30°C. Reduction of the periodate treated enzyme did not reverse the loss of stability. Binding of the synthetic substrate,p-nitrophenyl-α-D-glucoside, perturbed the environment around aromatic amino acids and caused a decrease in the ordered structure.

Isolation and characterization of the 7S protein from glanded cottonseed (Gossypium herbacium).

The major protein from glanded cottonseed has been isolated in a homogeneous form. Its S20, w value at 1% protein concentration is 6S in 1 Μ NaCl solution. It contains 1% carbohydrate and is free from phosphorus, gossypol (bound or free) and nucleic acid impurities. It consists of atleast seven non-identical subunits. The protein has an ultraviolet absorption maximum at 278 nm and fluorescence excitation and emission maxima at 280 nm and 325 nm respectively. Optical rotatory dispersion and circular dichroism measurements indicate that the protein consists predominantly of β-structure and random coil. The observed near-ultraviolet circular dichroic bands can be attributed to tyrosine, phenylalanine and tryptophan residues of the protein.