Production, Distribution, and Kinetic Properties of Inulinase in Continuous Cultures of Kluyveromyces marxianus CBS 6556

From a screening of several Kluyveromyces strains, the yeast Kluyveromyces marxianus CBS 6556 was selected for a study of the parameters relevant to the commercial production of inulinase (EC 3.2.1.7). This yeast exhibited superior properties with respect to growth at elevated temperatures (40 to 45°C), substrate specificity, and inulinase production. In sucrose-limited chemostat cultures growing on mineral medium, the amount of enzyme decreased from 52 U mg of cell dry weight⁻¹ at D = 0.1 h⁻¹ to 2 U mg of cell dry weight⁻¹ at D = 0.8 h⁻¹. Experiments with nitrogen-limited cultures further confirmed that synthesis of the enzyme is negatively controlled by the residual sugar concentration in the culture. High enzyme activities were observed during growth on nonsugar substrates, indicating that synthesis of the enzyme is a result of a derepression/repression mechanism. A substantial part of the inulinase produced by K. marxianus was associated with the cell wall. The enzyme could be released from the cell wall via a simple chemical treatment of cells. Results are presented on the effect of cultivation conditions on the distribution of the enzyme. Inulinase was active with sucrose, raffinose, stachyose, and inulin as substrates and exhibited an S/I ratio (relative activities with sucrose and inulin) of 15 under standard assay conditions. The enzyme activity decreased with increasing chain length of the substrate.     

Acyl coenzyme A:cholesterol acyltransferase in neonatal chick brain

An acyl coenzyme A:cholesterol acyltransferase activity which directly incorporates palmitoyl coenzyme A into cholesterol esters using endogenous cholesterol as substrate was demonstrated in microsomal preparations from neonatal chick brain. The enzyme showed, at pH 7.4, about 2-fold greater activity than that observed at pH 5.6. Nearly 10-times higher esterifying activity was found in brain microsomes using palmitoyl coenzyme A than that with palmitic acid. The acyltransferase activity was clearly different from the other cholesterol-esterifying enzymes previously found in brain, which incorporated free fatty acids into cholesterol esters and did not require ATP or coenzyme A as cofactors. Chick brain microsomes also incorporated palmitoyl coenzyme A into phospholipids and triacylglycerols. However, most of the radioactivity from this substrate was found in the fatty acid fraction, due to the presence of an acyl coenzyme A hydrolase activity in the enzyme preparations. Therefore, the formation of palmitate was tested during all the experiments. The brain acyltransferase assay conditions were optimized with respect to protein concentration, incubation time and palmitoyl coenzyme A concentration. Microsomal activity was independent of the presence of dithiothreitol in the incubation medium and microsomes can be stored at -40 degrees C for several weeks without losing activity. Addition of fatty acid-free bovine serum albumin to brain microsomal preparations produced a considerable increase in the acyltransferase activity, while acyl coenzyme A hydrolase was clearly inhibited. Results obtained show the existence in neonatal chick brain of an acyl coenzyme A:cholesterol acyltransferase activity similar to that found in a variety of tissues from different species but not previously reported in brain.     

Photo-CIDNP nuclear magnetic resonance as a probe for conformational changes in epidermal growth factor

Photo-chemically induced dynamic nuclear polarization (CIDNP)-NMR spectroscopy at 360 MHz has been used to investigate pH-induced conformational transitions in mouse epidermal growth factor. At about pH 9, all five tyrosine residues and both tryptophan residues are, to various extents, solvent-exposed, while the His-22 residue is buried in the protein matrix. Tyr-13 is the least exposed of the tyrosine residues and also the most immobilized. As the pH is decreased to 5.9, the tryptophan residues gradually become less exposed, while the Tyr-13 residue becomes internalized in the protein. These data suggest that the C-terminus and part of the N-terminal structural domain are affected by a conformational transition in mouse epidermal growth factor occurring between pH 6 and 8 via breakage of the His-22 inter-residue linkage. Above pH 9, a decreased photo-CIDNP effect is evident for both tryptophans and for Tyr-10 and Try-13; this information suggests that a second conformational change takes place at basic pH, which may simply be incipient denaturation.     

Identification of monoclonal antibodies specific for the T cell receptor complex by Fc receptor-mediated CTL lysis

Monoclonal antibodies (mAb) directed at the T cell receptor complex (TcR) on cloned T cells have generally been identified by their ability to inhibit the clone's antigen-specific function. Because such inhibition is highly dependent on antibody concentration and affinity, detection of anti-clonotypic antibodies to murine alloreactive T cells has been very difficult. In this report, an alternative method is described on the basis of the ability of antibodies specific for the TcR complex to activate T cells in an antigen-independent manner. The assay is based upon the observation that soluble antibodies to human T3 promote lysis of irrelevant, Fc receptor-positive targets by a human CTL line. By using this approach, an anti-TcR mAb has been identified among a panel of murine mAb generated against an alloreactive CTL clone. Induction of lysis by soluble anti-TcR mAb has been shown to require both the expression of Fc receptors on the target cell and conjugate formation between the effector and the target cell. This assay provides a screening procedure that is much more sensitive than inhibition of function, and it preferentially detects antibodies specific for cell surface molecules involved in T cell activation.     

Role of mevalonate-5-pyrophosphate decarboxylase in the regulation of chick intestinal cholesterogenesis

The response to different dietary conditions of the enzymes responsible for the transformation of mevalonic acid to isopentenyl pyrophosphate has been studied for the first time in the small bowel of the chick to elucidate the role of these enzymes in the regulation of intestinal cholesterogenesis. Feeding a 2% cholesterol diet from hatching resulted in a small but significant inhibition of mevalonate-5-pyrophosphate decarboxylase, while mevalonate kinase and mevalonate-5-phosphate kinase remained unaltered. Similar results were obtained for the three enzymes when 13-day-old chicks fed a standard fat-free diet were switched to a 5% cholesterol diet. Starved chicks exhibited lower intestinal decarboxylase activity than chicks fed a standard diet, while refeeding resulted in levels of activity similar or slightly greater than controls. None of the enzymes effecting the conversion of mevalonate to isopentenyl pyrophosphate in the small intestine presented diurnal variations. Results obtained suggest that mevalonate-5-pyrophosphate decarboxylase may play a significant role in the regulation of cholesterol synthesis in the small intestine.     

Stereometry in very close-range stereophotogrammetry with non-metric cameras for human movement analysis

In this paper a stereophotogrammetric algorithm based on a black-box approach to the modelling of object to image spaces relationship is proposed. The algorithm is well suited for 'very close-range photogrammetry', with respect to experiments in which the measurement field is 0.5 X 0.5 X 0.5 m or smaller, as in the analysis of a few or small body segments movements. The attainable accuracy is high, better than 0.1% of the observation distance. Non-professional and even different cameras can be used. Consequently an inexpensive experimental set-up can be realized. A very simple, cheap and easily usable calibration object is needed. Computation time for the reconstruction of object-space co-ordinates of point body landmarks is one order of magnitude lower than in the case of the Direct Linear Transformation (DLT) (Abdel Aziz and Karara, Proceedings of the ASP/U1 Symposium on Close-Range Photogrammetry, pp. 1-18. American Society of Photogrammetry, 1971; Marzan and Karara, Proceedings of the Symposium on close-range Photogrammetric Systems, pp. 420-467. American Society of Photogrammetry, 1975). Computation time for calibration is two-fold in respect of the DLT. An example of application to the recording of the movements of the index finger with respect to the metacarpophalangeal joint is given.     

Evidence for titratable gating charges controlling the voltage dependence of the outer mitochondrial membrane channel,VDAC

Summary A voltage-dependent anion-selective channel, VDAC, is found in outer mitochondrial membranes. VDAC's conductance is known to decrease as the transmembrane voltage is increased in either the positive or negative direction. Charged groups on the channel may be responsible for this voltage dependence by allowing the channel to respond to an applied electric field. If so, then neutralization of these charges would eliminate the voltage dependence. Channels in planar lipid bilayers which behaved normally at pH 6 lost much of their voltage dependence at high pH. Raising the pH reduced the steepness of the voltage dependence and raised the voltage needed to close half the channels. In contrast, the energy difference between the open and closed state in the absence of a field was changed very little by the elevated pH. The groups being titrated had an apparent pK of 10.6. From the pK and chemical modification, lysine epsilon amino groups are the most likely candidates responsible for VDAC's ability to respond to an applied electric field.     

Channel formation in phospholipid bilayer membranes by the toxin ofHeminthosporium maydis,race T

Summary Southern Corn Leaf Blight is caused by a toxin produced by a virulent form ofHelminthosporium maydis (Race T). The toxin has been shown to uncouple oxidative phosphorylation and dissipate Ca²⁺ gradients in mitochondria isolated from susceptible, but not resistant, corn. The possibility that the toxin acted by increasing the permeability of membranes to ions was tested using a planar bilayer membrane system. Addition of the toxin to the bilayer system, under voltage-clamp conditions, resulted in stepwise increases in current across the phospholipid bilayer, a response characteristic for channel formers. Single-channel conductance in 1m KCl is 27 pS which corresponds to 1.7×10⁷ ions sec^–1 channel^–1 at 100 mV applied potential. The toxin channels are: (i) fairly uniform in conductance, (ii) ideally selective for K⁺ over Cl^–, and (iii) most conductive to H⁺. The channel showed the following selectivity for alkali metal cations: Rb⁺>K⁺>Cs⁺>Na⁺>Li⁺ (169731) based on the most frequently observed conductance in 1m chloride salts. The toxin showed no voltage dependence over the range of –100 to +100 mV. Channel formation was also a property of a synthetic analog (Cmpd IV) of the toxin. The ability of the native toxin to form channels may be a mode of toxin action on mitochondrial membranes from susceptible corn.