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-' at D = 0.1 h-l to 2 U mg of cell dry weight1 l at D = 0.8 h-'. 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.

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The membrane protein cytochrome b5 and the polar and hydrophobic fragments into which it is cleaved by trypsin have been investigated, with major emphasis on the deoxycholate-solubilized form of the protein. Molecular weight measurements show that both the intact protein and the fragments are in a monomeric state in deoxycholate and that a small peptide of perhaps 15 residues is excised when the fragments are formed. Measurements of Stokes radius show that the major fragments are globular, but that intact cytochrome b5 has an asymmetric shape, consistent with a structure composed of two globular domains joined by a link region that may be as long as 30 to 40 A. Circular dichroism measurements were made in the far-ultraviolet and in the Soret region, and they add to previously existing data to make it virtually certain that the polar heme-containing domain is unaffected by proteolysis or by removal of deoxycholate. A significant change in the ultraviolet circular dichroism is, however, observed when proteolysis occurs and it is likely that it arises from the link between the domains, which appears to be highly structured (perhaps helical) in the intact protein, but randomly coiled after it is excised. The binding studies reported previously from this laboratory suggest that these inferences about the structure of cytochrome b5 in deoxycholate solution apply also to the protein as solubilized by detergent micelles, by phospholipid vesicles, or by the microsomal membrane.

The membrane toxin V"2 from the venom of Naja mossamhica niossambica was investigated in aqueous solution by one-dimensional and two-dimensional high-resolution nuclear magnetic resonance (NMR) techniques at 360 MHz. The spectral characterization included identification of the complete spin systems for several amino acid residues, nuclear Overhauser effect measurements, the use of chemically induced dynamic nuclear polarization and studies of the pH dependence of the N M R spectrum. Data from homologous toxins, in particular direct lytic factor 12B from Haemachatus haenzachatus, were used to establish assignments of aromatic and methyl proton resonances. From these experiments a short, triple-stranded fragment of antiparallel p structure could be determined, which includes the residues 23 -27, 43 -46 and 60 -62. Furthermore, the nuclear Overhauser effect measurements indicate close proximity in the protein conformation of the aromatic rings of Trp-14, Tyr-25 and Tyr-59, and the side chain of Ile-46.Keen interest in the toxins from the venoms of snakes belonging to the family Elapidae (cobra, coral snake, sea snake, etc.) has generated an extensive literature [l -31. Three classes of toxins are usually distinguished: long neurotoxins, short neurotoxins, and membrane toxins. These three classes form a group of homologous proteins with 71 -74, 60 -62, and 60-62 amino acid residues, respectively, arranged in a single polypeptide chain. If one excludes an extra disulfide bridge in the long neurotoxins, all of the approximately 80 toxins sequenced so far have four disulfide bridges linking half-cysteine residues which have homologous locations in the sequence. In addition seven other amino acid residues are located at homologous positions. Within a class or subclass of toxins, homology is even more extensive and can be as high as 60-70 % (e.g. membrane toxins from the genus Nuja). In spite of the extensive homology, there are pronounced differences in the mode of action of the different types of toxins. Neurotoxins bind to a protein receptor at the post-synaptic level and block acetylcholine reception [4]. The action of membrane'toxins results in a variety of effects including hemolysis, cytotoxieity, depolarization of excitable membrane and modulation of membranal enzyme activity [2,5]. This varied phenomenology has produced a profusion of trivial names such as cardiotoxin, cytotoxin, and direct lytic factor. A common trait of the actions of the different membrane toxins at the cellular level appears to be binding to the cell membrane with disturbance of its organization and function [5 -71.A variety of approaches have been used to investigate the spatial structures of snake neurotoxins and cardiotoxins [I, 21. These have included structure-activity modelling studies employing in part Chou-Fasman-type predictions [8 -lo], Abbreviations. CD, circular dichroism; 6, chemical shift; lytic factor 12B, direct lytic factor 12B from Haemachatus haemachaius; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; ppi...

A quantitative study of the changes in the protein pattern of the salivary glands of female Rhipicephalus evertsi evertsi during the entire repletion process was undertaken. These results, in conjunction with the previously determined toxic phase, indicated the presence of a toxic protein. The development of a sensitive in vitro assay using a Xenopus nerve-muscle preparation, made it possible to identify toxic phases during feeding and to assay fractions of salivary gland extracts during toxin isolation. Sufficient amounts of electrophoretically and chromatographically homogeneous toxin could be obtained through the use of chromatofocusing, enabling its characterization with respect to molecular weight (68 kDa; determined by gel permeation chromatography), pI (6.00), and amino acid composition. The toxin was inactivated by pronase digestion as well as by antiserum.