Some kinetic properties of the β- -glucosidase (cellobiase) in a commercial cellulase product from Penicillium funiculosum and its relevance in the hydrolysis of cellulose

Some kinetic parameters of the β- -glucosidase (cellobiase, β- -glucoside glucohydrolase, EC 3.2.1.21) component of Sturge Enzymes CP cellulase [see 1,4-(1,3;1,4)-β- -glucan 4-glucanohydrolase, EC 3.2.1.4] from Penicillium funiculosum have been determined. The Michaelis constants (K_m) for 4-nitrophenyl β- -glucopyranoside (4NPG) and cellobiose are 0.4 and 2.1 mM, respectively, at pH 4.0 and 50°C. -Glucose is shown to be a competitive inhibitor with inhibitor constants (K_i) of 1.7 mM when 4NPG is the substrate and 1 mM when cellobiose is the substrate. Cellobiose, at high concentrations, exhibits a substrate inhibition effect on the enzyme. -Glucono-1,5-lactone is shown to be a potent inhibitor (K_i = 8 μM; 4NPG as substrate) while -fructose exhibits little inhibition. Cellulose hydrolysis progress curves using Avicel or Solka Floc as substrates and a range of commercial cellulase preparations show that CP cellulase gives the best performance, which can be attributed to the activity of the β- -glucosidase in this preparation in maintaining the cellobiose at low concentrations during cellulose hydrolysis.     

Behavior,net reproduction,longevity, and mummy-stage survival ofAphidius matricariae [Hym. Aphidiidae]

The courtship, mating and ovipositional behavior ofA. matricariae Haliday were studied. UsingMyzus persicae (Sulzer) as the host, the production of progeny per female parasite and survival from mummy stage to the adult were studied at constant temperatures of 10°, 12.8°, 15.6°, 18.3°, 21°, 24°, 26.7°, 29.5° and 32°C. The longevity of male and female parasites was determined at temperatures of 7°, 10°, 15.6°, 21°, 26.7°, 29.5° and 32°C. The greatest number of progeny (392) was produced at 21°C. The optimal temperatures for production of progeny and survival of the parasites during the mummy stage were from 12.8°C to 21°C. The longevity of male and female adult parasites decreased as temperatures increased and male parasites lived significantly (P<0.05) longer than females at 10° and 15.6°C.     

Pineal cells enhance choline acetyltransferase activity in sympathetic neurons

Cells derived from the neonatal rat pineal gland were cocultured with cells derived from neonatal rat superior cervical ganglia (SCG) in an attempt to determine whether a sympathetic target organ with only adrenergic properties could enhance the development of adrenergic transmitter properties in sympathetic neurons in tissue culture. Choline acetyltransferase was measured as an index of cholinergic differentiation, and tyrosine hydroxylase was measured as an index of adrenergic differentiation. As indices of total cell number and cellular volume, DNA and protein, respectively, were also measured. We found that the pineal-SCG cocultures contained ten times greater choline acetyltransferase activity than sister neuronal cultures cultured without pineal cells, thus indicating that the pineal cells enhanced cholinergic properties in the sympathetic neurons. This cholinergic enhancement was dependent upon the presence of nerve growth factor and could not be obtained with pineal-conditioned medium. Tyrosine hydroxylase activity, measured on cultures sister to those mentioned above, was low in all cultures and decreased somewhat in SCGs cultured alone. TH activity in the pineal-SCG cocultures, however, increased slightly. Some tyrosine hydroxylating activity developed in pineals cultured alone, however, and may have been responsible for the small increase in tyrosine hydroxylase activity noted in the pineal-SCG cocultures. The implications of these results for a determination of the role that target organ plays in the development of the transmitter properties of sympathetic neurons are discussed.     

Fb''2, a new peptic fragment of human immunoglobulin G.

The digestion of a human IgG1 K myeloma protein with pepsin in the presence of 8M-urea was observed to produce a fragment, designated Fb'2, which differed from the products of aqueous peptic digestion and from other characteristic immunoglobulin digestion products. 2. Fragment Fb's was also found when two other IgG1/K proteins were treated similarly. 3. Sedimentation-equilibrium studies showed the mol.wt. of fragment Fb'2 to be 56800. 4. On reduction, two equivalents of each of three peptides were released from fragment Fb's; these were characterized by N- and C-terminal determinations and by amino acid sequencing. 5. Fragment Fb'2 was shown to consist of the constant regions of both light chains, from residue Ile-117 to the C-terminus, and the CH1 domains and hinge region of the heavy chains, from residue Val-113 to residue Met-252, with a gap of five residues within the intrachain disulphide loop, between residues Leu-174 and Tyr-180.     

Subunit dissociation and unfolding of rabbit muscle phosphofructokinase by guanidine by hydrochloride.

The denaturation of rabbit skeletal muscle phosphofructokinase by guanidine hydrochloride has been studied using fluorescence, light scattering, and enzyme activity measurements. The transition from fully active tetramer (0.1 M potassium phosphate (pH 8.0) at 10 and 23 degrees) to unfolded polypeptide chains occurs in two phases as measured by changes in the fluorescence spectrum and light scattering of the protein: dissociation to monomers at low guanidine hydrochloride concentrations (similar to 0.8 M) followed by an unfolding of the polypeptide chains, which presumably results in a random coil state, at high concentrations of denaturant (greater than 3.5 M). The initial transition can be further divided into two distinct stages. The native enzyme is rapidly dissociated to inactive monomers which then undergo a much slower conformational change that alters the fluorescence spectrum of the protein. The dissociation is complete within 2 min and is reversible, but the conformational change requires about 2 hr for completion and is not reversible under a variety of conditions, including the presence of substrates and allosteric effectors. The conformationally altered protomer reaggregates to form a precipitate at 23 degrees, but is stable below 10 degrees. The second major phase of the denaturation is fully reversible. A simple mechanism is proposed to account for the results, and its implications for the corresponding renaturation process are discussed.     

A purification procedure for the soluble cytochrome oxidase and some other respiratory proteins from Pseudomonas aeruginosa.

The production of the soluble cytochrome oxidase/nitrite reductase in the bacterium Pseudomonas aeruginosa is favoured by anaerobic conditions and the presence of KNO3(20g/l) in the culture medium. Of three methods commonly used for the disruption of bacterial suspensions (ultrasonication, liquid-shear homogenization and glass-bead grinding), sonication proved the most efficient in releasing the Pseudomonas cytochrome oxidase. A polarographic assay of Pseudomonas cytochrome oxidase activity with sodium ascorbate as substrate and NNN'N'-tetramethyl-p-phenylenediamine dihydrochloride as electron mediator is described. A purification procedure was developed which can be used on the small scale (40-litre cultures) or the large scale (400-litre cultures) and provides high yields of three respiratory-chain proteins, Pseudomonas cytochrome oxidase, cytochrome c551 and azurin, in a pure state. A typical preparation of 250g of Ps.aeruginosa cell paste yielded 180mg of Pseudomonas cytochrome oxidase, 81 mg of Pseudomonas cytochrome c551 and 275mg of Pseudomonas azurin.     

A technique for the determination of enantiomeric amino acids in biological samples

Racemic amino acids can be separated into their enantiomers by means of gas-liquid chromatography. The most applied technique, today, is the conversion of chiral compounds into diastereoisomers with optically active reagents and subsequent chromatography on conventional optically inactive stationary phases. In previous studies it has been realized that this technique is associated with various problems. We studied the use of optically active stationary phases for separating enantiomers directly via a diastereoisomeric association complex. The optically active stationary phases employed are N- and C-terminal substituted dipeptides of the type N-trifluoroacetyl-dipeptide-cyclohexyl esters and have been synthesised by the I-hydroxibenztriazole dicyclohexylcarbodiimide method. The quality of these phases with respect to separation factors, resolution factors, and thermodynamical properties have been evaluated. All synthetic phases show excellent properties; however, when attempting separation of mixtures of naturally occurring amino acids extensive overlap in the elution diagram was detected. Only one phase — N-TFA-L-α-amino-n-butyryl-L-α-amino butyric acid cyclohexyl ester gave complete resolution of the naturally occurring amino acids alanine, valine, glycine, threonine, eucine, isoleucine, serine and proline on a 400 ft × 0.02 in capillary column. Less volatile amino acids such as aspartic acid, phenylalanine, methionine, glutamic acid, tyrosine, arginine, and tryptophan can be resolved at a 100 ft×0.02 in column.     

Geographical variation in greater covert moult in first winter Coal Tits Parus ater

The state of greater covert moult in 145 first winter Coal Tits Parus ater from 5 suburban English sites was examined. There was a statistically significant difference between sites in the number of greater coverts moulted, with a tendency for birds from more northerly and easterly sites to have more old, unmoulted greater coverts than birds from more southerly sites. The results could not be fully explained by variations between seasons in the number of greater coverts moulted or in the relative proportions of birds from second broods. The possible effects of habitat differences are discussed and it is suggested that birds from more northerly areas may moult fewer greater coverts as an energy conservation measure.