Purification and characterization of α-glucosidases produced by Saccharomyces in response to three distinct maltose genes

α-Glucosidases or maltases (EC 3.2.1.20) were purified to electrophoretic homogeneity from a respective strain of Sacchromyces cerevisiae which carries a single MAL gene, either MALα, MALβ or MALγ, using gluconate-Sepharose affinity chromography and isoelectrofocusing. Of these maltases, two types of maltase were obtained from the MALγ strain, the pI values of which were 5.6 and 5.9. From the MALα and MALβ strain was obtained only one type of maltase with the pI at 5.6 which was identical to one of the maltases from the MALγ strain. These four maltases possessed the same properties, except for pI. They were monomers with molecular weights of between 66 000 and 67 000. With regard to the substrate specificity, they hydrolyzed maltose and sucrose exclusively but not α-methulglucoside nor maltooligosaccharide. They did not differ in immunological properties.     

Isolation and Characterization of a Catabolite Repression-Insensitive Mutant of a Methanol Yeast, Candida boidinii A5, Producing Alcohol Oxidase in Glucose-Containing Medium

Mutants exhibiting alcohol oxidase (EC 1.1.3.13) activity when grown on glucose in the presence of methanol were found among 2-deoxyglucose-resistant mutants derived from a methanol yeast, Candida boidinii A5. One of these mutants, strain ADU-15, showed the highest alcohol oxidase activity in glucose-containing medium. The growth characteristics and also the induction and degradation of alcohol oxidase were compared with the parent strain and mutant strain ADU-15. In the parent strain, initiation of alcohol oxidase synthesis was delayed by the addition of 0.5% glucose to the methanol medium, whereas it was not delayed in mutant strain ADU-15. This showed that alcohol oxidase underwent repression by glucose. On the other hand, degradation of alcohol oxidase after transfer of the cells from methanol to glucose medium (catabolite inactivation) was observed to proceed at similar rates in parent and mutant strains. The results of immunochemical titration experiments suggest that catabolite inactivation of alcohol oxidase is coupled with a quantitative change in the enzyme. Mutant strain ADU-15 was proved to be a catabolite repression-insensitive mutant and to produce alcohol oxidase in the presence of glucose. However, it was not an overproducer of alcohol oxidase and, in both the parent and mutant strains, alcohol oxidase was completely repressed by ethanol.     

Determination of free sulphite in wine using a microbial sensor

Summary A microbial sensor of immobilized Thiobacillus thiooxidans S3 cells was assembled to determine free sulphite in wine. Sulphite oxidation activity of the immobilized cells was sufficiently high for use even after 3 months storage at 4° C. The sensitivity of this sensor was 116 nA·1·mg^-1 for sulphur dioxide. The relationship between the current decrease and the sulphur dioxide concentration was linear up to 17 mg·1^-1. The sampling rate achieved was 10 min per sample including washing time. This sensor method needed no pretreatment of wine samples, and wines diluted with 5 mM sulphuric acid solution could be directly introduced in the computer-aided analysis system. The pigments in red wine did not disturbed the analysis.     

The usefulness of CHAPS as a non-cytotoxic stabilizing agent in purification of growth factors

Among several detergents, a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), was found to be least cytotoxic for cultured mammalian cells. CHAPS improved the activity recovery and elution profile of crude and purified fibroblast growth factors (FGFs) during chromatographies. Diluted preparations of FGFs were stabilized by CHAPS against the loss during storage. Amino acid sequence analysis was not disturbed by CHAPS. CHAPS was removable by reversed-phase high-performance liquid chromatography. These results indicate that CHAPS is useful as a non-cytotoxic stabilizing agent in purification of various kinds of bioactive polypeptides.Abbreviations -MEM Alpha Modification of Eagle's Minimal essential medium - CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate - CHAPSO 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate - CS Calf Serum - EGF Epidermal Growth Factor - FGF Fibroblast Growth Factor - HPLC High-Performance Liquid Chromatography - NGF Nerve Growth Factor - NOG 1-O-n-octyl--D-glucopyranoside - NP-40 Nonidet P-40 - PBS Phosphate-Buffered Saline - SB 12 3-(dodecylmethylammonio)-1-propane sulfonate - SDS Sodium Dodecyl Sulfate - TGF- and Transforming Growth Factor type and      

Heterogeneous distribution of the precursor of type I and type III collagen and fibronectin in the rough endoplasmic reticulum of palatal mesenchymal cells of the mouse embryo cultured in ascorbate-depleted medium

Summary In order to examine the intracellular distribution of precursors of type I and type III collagen and fibronectin in the palatal mesenchymal (MEPM) cells of the mouse embryo cultured under ascorbate-deficient conditions, immuno-electron-microscopic studies were carried out by use of affinity purified antibodies for these proteins. MEPM cells were obtained from the palatal shelves of 14-day-old mouse fetuses and cultured for 3–7 days in medium, either with or without 50 ng/dish/day ascorbic acid. Results obtained were as follows: (1) Although the rough endoplasmic reticulum (rER) of MEPM cells cultured for 5 days in ascorbate-supplemented medium was flattened, that in cells cultured in ascorbate-deficient medium had a distended or vesicular appearance. (2) Vesicular or distended rER showed heterogeneous staining for both type I and type III collagen, namely, some parts of rER showed positive staining for both types of collagen, while others showed negative staining. (3) Both type I and type III collagen showed codistribution in the same vesicular rER. (4) Vesicular rER showed negative or very faint labelling for fibronectin. These results may suggest regional differences in the function of rER.     

Effects of the Cortex on Light- and Kinetin-Induced Accumulation of Betacyanin in the Epidermal Tissue of Phytolacca americana

Accumulation of betacyanin in the peeled green epidermis fromthe stem of P. americana was induced by incubating the epidermisin Murashige and Skoog's medium, under light, and was promotedby the presence of kinetin. However, in the epidermal tissuewith cortex attached, the accumulation of betacyanin was inhibited. (Received March 27, 1989; Accepted January 24, 1990)     

Enzymatic synthesis of alkyl xylobioside and xyloside from xylan and alcohol

Summary Alkyl -D-xylobioside and alkyl -D-xyloside were prepared by the one-pot reaction of xylan and a fatty alcohol, such as 1-octanol, 1-decanol, 2-octanol and 2-ethylhexanol using the cell-free culture filtrate of the xylan-assimilating strain, Aureobasidium pullulans KK415. Using this strain, a novel surfactant, alkyl -D-xylobioside, was produced as the main product when the alcohol and xylan was incubated at a temperature of 65 °C and pH 4.5.     

Dopamine-Releasing Action of 6R-l-erythro-Tetrahydrobiopterin: Analysis of Its Action Site Using Sepiapterin

Abstract: Recently, we reported that 6 R - l - erythro -tetrahydrobiopterin (6 R -BH₄), a natural cofactor for hydroxylases of tyrosine and tryptophan, has a monoamine-releasing action independent of its cofactor activity. Here we attempted to determine whether 6 R -BH₄ acts inside the cell or from the outside of the cell by using brain microdialysis in the rat striatum. For this purpose, sepiapterin, an immediate precursor of 6 R -BH₄ in the salvage pathway, was used to selectively increase the intracellular 6 R -BH₄ levels. Dialytic perfusion of sepiapterin increased tissue levels of reduced biopterin (mainly 6 R -BH₄) but not the extracellular levels. Administration of sepiapterin increased the extracellular levels of 3,4-dihydroxyphenylalanine (DOPA) (an index of in vivo tyrosine hydroxylase activity) and of dopamine (DA) (an index of in vivo DA release). Either of the increases was eliminated after pretreatment with a tyrosine hydroxylase inhibitor α-methyl- p -tyrosine. Administration of 6 R -BH₄ increased extracellular levels of reduced biopterin, DOPA, and DA. After pretreatment with α-methyl- p -tyrosine, the increase in DOPA levels was abolished, but most of the increase in DA levels persisted. The increase in DA levels also persisted after pretreatment with nitric oxide synthase inhibitors. These data demonstrate that 6 R -BH₄ stimulates DA release directly, independent of its cofactor action for tyrosine hydroxylase and nitric oxide synthase, by acting from the outside of neurons.     

Integrated tandem affinity protein purification using the polyhistidine plus extra 4 amino acids (HiP4) tag system

Peptide tag systems are a robust biophysical and biochemical method that is widely used for protein detection and purification. Here, we developed a novel tag system termed “HiP4” (histidine plus four amino acids) whose epitope sequence comprises only seven amino acids (HHHDYDI) that partially overlap with the conventional 6x histidine tag (6xHis-tag). We produced a monoclonal antibody against the HiP4 tag that can be used in multiple immunoassays with high specificity and affinity. Using this system, we developed a tandem affinity purification (TAP) and mass spectrometry (TAP-MS) system for comprehensive protein interactome analysis. The integrated use of nickel bead purification followed by HiP4 tag immunoprecipitation made it possible to reduce nonspecific binding and improve selectivity, leading to the recovery of previously unrecognized proteins that interact with hepatitis B virus X (HBx) protein or TAR DNA-binding protein 43 (TARDBP or TDP-43). Our results indicate that this system may be viable as a simple and powerful tool for TAP-MS that can achieve low background and high selectivity in comprehensive protein–protein interaction analyses.