Wilfred Niels Arnold

β-fructofuranosidase from grape berries

Summary 1. An enzyme from mature grape berries that hydrolyzes sucrose was purified 55-fold on a protein basis and 135-fold on a polysaccharide basis. 2. The substrate specificity was consistent with that of a β-fructofuranosidase (β- D -fructofuranoside fructohydrolase, EC 3.2.1.26). 3. At pH 4 for sucrose Km = 4 mM, relative maximum velocity = 100; for raffmose Km = 22 mM, relative maximum velocity = 47; and for methyl β- D -fructofuranoside Km = 167 mM, relative maximum velocity = 77. 4. Maximum velocity for sucrose approaches a maximum at pH 2. Km changes continuously from 19 mM at pH 2 to 0.8 mM at pH 5.85. 5. Purified grape β-fructofuranosidase is stable at 30° from pH 2 to pH 7. At 3° under toluene and at pH 4 it is stable for several weeks. At 85° inactivation is rapid and irreversible. Heavy-metal ions (especially Hg2+, Pb2+, UO22+, and Ag+) and aniline are inhibitors. 6. Sumner s9 3,5-dinitrosalicylic acid reagent in conjunction with semiautomatic syringes was found to be a convenient assay procedure for large numbers of incubations.

Porphyrogenic properties of the terpenes camphor, pinene, and thujone (with a note on historic implications for absinthe and the illness of Vincent Van Gogh)

Camphor, alpha-pinene (the major component of turpentine), and thujone (a constituent in the liqueur called absinthe) produced an increase in porphyrin production in primary cultures of chick embryo liver cells. In the presence of desferrioxamine (an iron chelator which inhibits heme synthesis and thereby mimics the effect of the block associated with acute porphyria), the terpenes enhanced porphyrin accumulation 5- to 20-fold. They also induced synthesis of the rate-controlling enzyme for the pathway, 5-aminolevulinic acid synthase, which was monitored both spectrophotometrically and immunochemically. These effects are shared by well-known porphyrogenic chemicals such as phenobarbital and glutethimide. Camphor and glutethimide alone led to the accumulation of mostly uro- and heptacarboxylporphyrins, whereas alpha-pinene and thujone resulted in lesser accumulations of porphyrins which were predominantly copro- and protoporphyrins. In the presence of desferrioxamine, plus any of the three terpenes, the major product that accumulated was protoporphyrin. The present results indicate that the terpenes tested are porphyrogenic and hazardous to patients with underlying defects in hepatic heme synthesis. There are also implications for the illness of Vincent van Gogh and the once popular, but now banned liqueur, called absinthe.

The selection of sucrose as the translocate of higher plants

Abstract In the absence of a successful physical model to explain the choice of sucrose as the translocate of higher plants, the author advances the thesis that sucrose acts as a protected derivative of glucose. According to this hypothesis, the requisites of a good translocate include that it should be a non-reducing, easily hydrolyzed derivative of glucose. Various properties of the sucrose molecule enhance its suitability for such a role. The unusual translocates encountered in some species are more complex derivatives of glucose but include a sucrose residue in their structure.

Heat inactivation kinetics of yeast β-fructofuranosidase. A polydisperse system

Abstract 1. 1. The kinetics of heat inactivaton of yeast β-fructofuranosidase were studied at 65° in the presence of 10 mM acetate buffer (pH 5.0). 2. 2. The orders of reaction with respect to time ( n t ) and with respect to concentration ( n c ) were determined on a relatively pure enzyme preparation. It was found that n t > 1 and n c = 1. 3. 3. The same enzyme preparation was chromatographed on DEAE-cellulose with a linear gradient. The symmetrical elution pattern displayed almost constant specific activity on a protein basis. This represented only slight improvement only slight improvement on the starting specific activity. 4. 4. Individual chromatographic fractions gave n t = I. However, the magnitude of their first-order rate constants ( k ) increased continuously from the “front” to the “rear” of the peak of their first-order rate constants ( k ) increased continuously from the “front” to the “rear” of the peak. 5. 5. There was a trend in the polysaccharide content of chromatographic fractions from 54% at the “front” to 39% at the “rear”. 6. 6. It was concluded that this preparation of β-fructofuranosidase is polydisperse with respect to polysaccharide content and to heat-inactivation susceptibility. 7. 7. Heat inactivation of unfractionated enzyme is evidently a complex of simultaneous unimolecular processes. This is sufficient to explain the apparent anomaly that n t > 1 but n c = 1.

The Illness of Vincent van Gogh

Vincent van Gogh (1853–1890) was a wonderfully accomplished artist whose work is now widely appreciated. He created a great number of masterpiece paintings and drawings in just one decade devoted to art. His productivity is even more remarkable when considered in the context of his debilitating illness. He suffered from medical crises that were devastating, but in the intervening periods he was both lucid and creative. He left a profound, soul-searching description of his jagged life in his correspondence, which provides the basis for the present analysis. An inherited metabolic disease, acute intermittent porphyria, accounts for all of the signs and symptoms of van Gogh’s underlying illness. On this 150th anniversary of the birth of Vincent van Gogh it is appropriate to revisit the subject and to analyze the lack of organized skepticism in the popular media about other diagnoses.Vincent van Gogh (1853-1890) was a wonderfully accomplished artist whose work is now widely appreciated. He created a great number of masterpiece paintings and drawings in just one decade devoted to art. His productivity is even more remarkable when considered in the context of his debilitating illness. He suffered from medical crises that were devastating, but in the intervening periods he was both lucid and creative. He left a profound, soul-searching description of his jagged life in his correspondence, which provides the basis for the present analysis. An inherited metabolic disease, acute intermittent porphyria, accounts for all of the signs and symptoms of van Goghs underlying illness. On this 150th anniversary of the birth of Vincent van Gogh it is appropriate to revisit the subject and to analyze the lack of organized skepticism in the popular media about other diagnoses.

Purification and characterization of a dextranase from Sporothrix schenckii

Abstract A dextranase (EC 3.2.1.11) was purified and characterized from the IP-29 strain of Sporothrix schenckii, a dimorphic pathogenic fungus. Growing cells secreted the enzyme into a standard culture medium (20 °C) that supports the mycelial phase. Soluble bacterial dextrans substituted for glucose as substrate with a small decrease in cellular yield but a tenfold increase in the production of dextranase. This enzyme is a monomeric protein with a molecular mass of 79 kDa, a pH optimum of 5.0, and an action pattern against a soluble 170-kDa bacterial dextran that leads to a final mixture of glucose (38%), isomaltose (38%), and branched oligosaccharides (24%). In the presence of 200 mM sodium acetate buffer (pH 5.0), the Km for soluble dextran was 0.067 ± 0.003% (w/v). Salts of Hg2+, (UO2)2+, Pb2+, Cu2+, and Zn2+ inhibited by affecting both Vmax and Km. The enzyme was most stable between pH values of 4.50 and 4.75, where the half-life at 55 °C was 18 min and the energy of activation for heat denaturation was 99 kcal/mol. S. schenckii dextranase catalyzed the degradation of cross-linked dextran chains in Sephadex G-50 to G-200, and the latter was a good substrate for cell growth at 20 °C. Highly cross-linked grades (i.e., G-10 and G-25) were refractory to hydrolysis. Most strains of S. schenckii from Europe and North America tested positive for dextranase when grown at 20 °C. All of these isolates grew on glucose at 35 °C, a condition that is typically associated with the yeast phase, but they did not express dextranase and were incapable of using dextran as a carbon source at the higher temperature.

Selective Inactivation of an Extra-cytoplasmic Acid Phosphatase of Yeast-like Cells of Sporothrix schenckii by Sodium Fluoride

Suspensions of intact, yeast-like cells of Sporothrix schenckii exhibited an acid phosphatase (EC 3.1.3.2) activity against p-nitrophenyl phosphate of about 5 IU (g dry wt)-1, without recourse to membrane perturbation. This extra-cytoplasmic acid phosphatase was reversibly and competitively inhibited by orthophosphate (Ki = 2 mM at pH 5) but unaffected by L(+)-tartrate (in contradistinction to some of the cytoplasmic acid phosphatases of the same organism). Inactivation by NaF of the extra-cytoplasmic isoenzyme was irreversible and followed first order kinetics; sensitivity to NaF was decreased by the presence of citrate, phosphate or substrate. Neither Km (0.3 mM at pH 5) nor Vmax for this enzyme in acetate buffer was greatly affected by pH in the range 3-5 but the first order rate constant for inactivation by NaF was strongly dependent on pH (maximum at pH 3.5). Crude cell-free extracts of yeast cells had nine electrophoretically distinct acid phosphatase activity bands and, on the basis of the pattern of inhibitors, the extra-cytoplasmic activity was identified as Y-I, an isoenzyme that barely penetrates standard polyacrylamide gel electropherograms. Additional evidence for the assignment came from selective inactivation of this isoenzyme by short treatments of intact cells with NaF under conditions that did not allow penetration of the plasma membrane by the inhibitor and did not kill the cells.

Cytochemical localization of acid phosphatases in the dimorphic fungusSporothrix schenckii

A modified Gomori procedure at the electron microscopic level revealed a multiplicity of acid phosphatase activity sites in both yeast-like and mycelial phase cells. Vacuoles and the periplasmic space contained electron opaque deposits (lead phosphate) that were absent in control incubations either lacking the substrate (β-glycerophosphate) or fortified with an inhibitor (sodium fluoride). The outermost region of the cell envelope was also active and, in contradistinction to previous examples with other yeasts, deposition of lead phosphate in this locale occurred even when the rate of orthophosphate generation was drastically reduced by lowering the substrate concentration. When mechanically disrupted yeast-like cells were washed and then subjected to the cytochemical procedure, pieces of broken cell envelope gave a positive reaction. The reaction product was invariably restricted to one side of cell wall cross sections. A specific and novel association of acid phosphatase with a microfibrillar zone was indicated.

Selenium concentration in the prostate.

Samples of healthy tissue were taken from each of six prostatectomy specimens (55–72 yr), digested with acids, and analyzed for selenium by atomic fluorescence spectroscopy. The concentrations for five specimens were 1.32±0.09 µg Se/g dry wt (range 1.24–1.42) and 0.213±0.012 µg Se/g wet wt (range 0.200–0.229). The other specimen, from a 58-yr-old man who was the only one within this study to take a 200-µg Se/d dietary supplement, contained 2.72 µg Se/g dry wt and 0.421 µg Se/g wet wt.

Scanning electron microscopy of cells and protoplasts ofSaccharomyces rouxii

Cells ofSaccharomyces rouxii from a normal broth culture were subjected to a high osmotic pressure (2 M KCl), fixed in 3% glutaraldehyde fortified with 2 M KCl, and then processed routinely for examination in a scanning electron microscope. Micrographs revealed birth and bud scars typical for the genus and an apparently undamaged surface topography. Protoplasts were prepared from the same material by digestion of cell walls with snail gut enzymes in the presence of 2 M KCl. Naked protoplasts were obtained and these exhibited surface invaginations. In addition, spheroidal protrusions were noted and these structures were equated with the periplasmic bodies previously described by transmission electron microscopy. The propensity for periplasmic body formation inSaccharomyces rouxii is contrasted with otherSaccharomyces species and the circumstantial evidence that relates periplasmic bodies to cryptic β-fructofuranosidase inS. rouxii is briefly discussed.