Induction, isolation, and some properties of the NADPH-dependent glutamate dehydrogenase from the nonheterocystous cyanobacterium Phormidium laminosum.

The level of the NADPH-dependent glutamate dehydrogenase activity (EC 1.4.1.4) from nitrate-grown cells of the thermophilic non-N2-fixing cyanobacterium Phormidium laminosum OH-1-p.Cl1 could be significantly enhanced by the presence of ammonium or nitrite, as well as by L-methionine-DL-sulfoximine and other sources of organic nitrogen (L-Glu, L-Gln, and methylamine). The enzyme was purified more than 4,400-fold by ultracentrifugation, ion-exchange chromatography, and affinity chromatography, and at 30 degrees C it showed a specific activity of 32.9 mumol of NADPH oxidized per min per mg of protein. The purified enzyme showed no aminotransferase activity and catalyzed the amination of 2-oxoglutarate preferentially to the reverse catabolic reaction. The enzyme was very specific for its substrates 2-oxoglutarate (Km = 1.25 mM) and NADPH (Km = 64 microM), for which hyperbolic kinetics were obtained. However, negative cooperativity (Hill coefficient h = 0.89) and [S]0.5 of 18.2 mM were observed for ammonium. The mechanism of the aminating reaction was of a random type with independent sites. The purified enzyme showed its maximal activity at 60 degrees C (Ea = 5.1 kcal/mol [21.3 kJ/mol]) and optimal pH values of 8.0 and 7.5 when assayed in Tris hydrochloride and potassium phosphate buffers, respectively. The native molecular mass of the enzyme was about 280 kilodaltons. The possible physiological role of the enzyme in ammonia assimilation is discussed.     

Chitin synthetase activity is bound to chitosomes and to the plasma membrane in protoplasts of Saccharomyces cerevisiae

The sub-cellular distribution of chitin synthetase was studied in homogenates of Saccharomyces cerevisiae protoplasts. Use of a mild disruption method minimized rupture of vacuoles and ensuing contamination of subcellular fractions by vacuolar proteinases. After fractionation of whole or partially purified homogenates through an isopycnic sucrose gradient chitin synthetase activity was found to be distributed between two distinct particulate fractions with different buoyant density and particle diameter. When whole homogenates were used, about 52% of the chitin synthetase loaded was localized in a microvesicular population identified as chitosomes (diameter 40-110 nm; buoyant density (d) = 1.146 g/cm3). Another vesicular population containing 26% of the activity was identified as plasma membrane vesicles because of its large mean diameter (260 nm), its high buoyant density (d = 1.203 g/cm3) and by the presence of the vanadate-sensitive ATPase activity. Moreover, after surface labeling of protoplasts with 3H-concanavalin A, the label cosedimented with the presumed plasma membrane vesicles. There was a negligible cross-contamination of the chitosome fraction by yeast plasma membrane markers. In both the plasma membrane and the chitosome fractions, the chitin synthetase was stable and essentially zymogenic. Activation of the chitosome fraction produces microfibrils 100-250 nm in length. Our results support the idea that chitosomes do not originate by plasma membrane vesiculation but are defined sub-cellular organelles containing most of the chitin synthetase in protoplasts of Saccharomyces cerevisiae.     

Chemical intermediates in dopamine oxidation by tyrosinase,and kinetic studies of the process

A minor pathway for dopamine oxidation to dopaminochrome, by tyrosinase, is proposed. Characterization of intermediates in this oxidative reaction and stoichiometric determination have both been undertaken. After oxidizing dopamine with mushroom tyrosinase or sodium periodate in a pH range from 6.0 to 7.0, it was spectrophotometrically possible to detect o-dopaminoquinone-H⁺ as the first intermediate in this pathway. The steps for dopamine transformation to dopaminochrome are as follows: dopamine → o-dopaminequinone-H⁺ → o-dopaminequinone → leuko-dopaminochrome → dopaminochrome. No participation of oxygen was detected in the conversion of leukodopaminochrome to dopaminochrome. Scanning spectroscopy and graphical analysis of the obtained spectra also verified that dopaminequinone-H⁺ was transformed into aminochrome in a constant ratio. The stoichiometry equation for this conversion is 2 o-dopaminequinone-H⁺ → dopamine + dopaminochrome. The pathway for dopamine oxidation to dopaminochrome by tyrosinase has been studied as a system of various chemical reactions coupled to an enzymatic reaction. A theoretical and experimental kinetic approach is proposed for such a system; this type of mechanism has been named “Enzymatic-chemical-chemical” (E_ZCC). Rate constants for the implied chemical steps at different pH and temperature values have been evaluated from the measurement of the lag period arising from the accumulation of dopaminochrome that took place when dopamine was oxidized at acid pH. The thermodynamic activation parameters of the chemical steps, the deprotonation of dopaminequinone-H⁺ to dopaminequinone, and the internal cyclization of dopaminequinone to leukodopaminochrome have been calculated.     

The [Ca2+ + Mg2+]-dependent adenosine triphosphatase of SV40 transformed WI38 lung fibroblasts

The Ca2+-stimulated, Mg2+-dependent ATPase of SV40 transformed WI38 lung fibroblast homogenates exhibits a high affinity for Ca2+ (K0.5 = 0.20 microM) and moderately high affinity for ATP (Km = 28.6 microM) and Mg2+ (K0.5 = 138.5 microM). This activity was NaN3, KCN and oligomycin insensitive but very sensitive to vanadate (I50 = 0.5 microM) suggesting its being neither mitochondrial or microsomal but plasma membrane in origin. Under optimal conditions of protein, hydrogen ion and substrate concentration, 16-19 nmoles phosphate was released per min per mg protein. Hill plot analysis indicated no cooperativity to occur between Ca2+ binding sites. Nucleotides other than ATP and dATP were ineffective as substrates. The trivalent cation, lanthanum (La3+) completely inhibited hydrolysis of ATP at approximately 70 microM (I50 = 25 microM). Calmodulin antagonists trifluoperazine and calmidazolium inhibited ATP hydrolysis in a dose dependent fashion.     

The influence of PTH, calcium gluconate and EDTA on the parotid saliva in the goat

1. The role of exogenous parathyroid hormone (PTH) and stimulation or inhibition of endogenous hormone release, on the parotid gland of normal and thyroparathyroidectomized (t.x.p.t.x.) goats was studied. 2. The intravenous infusion of PTH and EDTA produced a transitory rise in saliva flow rate in intact animals. In t.x.p.t.x. goats the flow of saliva decreased transiently throughout the infusion. 3. The calcium levels in parotid saliva was unchanged throughout the infusion of PTH, EDTA, calcium gluconate both alone or with propranolol, in either intact or t.x.p.t.x. animals. 4. The parathyroid hormone infusion caused an increase in salivary phosphate concentration in both intact and operated goats. The effects of PTH upon the salivary flow and concentration of P are discussed.     

Quantitation of the β-bungarotoxin-induced release of lactate dehydrogenase from cerebrocortical synaptosomes

Treatment with -bungarotoxin for 1 h induces lactate dehydrogenase release from cerebrocortical synaptosomes. The effect is Ca²⁺-dependent as suggested by the inhibition observed with EGTA. An inhibition of the effect can be also obtained by addition of Sr²⁺ ions, suggesting that the phospholipase A₂ activity associated to the toxin is involved in the efflux of the enzyme. The -bungarotoxin-induced release of synaptoplasmic lactate dehydrogenase is a saturable effect, showing a half-maximal effect concentration of 32 nM and a maximal efflux of 25% of the total synaptosomal enzyme as calculated by double-reciprocal plot.     

The detection of linkage and heterogeneity in nuclear families for complex disorders: one versus two marker loci.

Using exact expected likelihoods, we have computed the average number of phase-unknown nuclear families needed to detect linkage and heterogeneity. We have examined the case of both dominant and recessive inheritance with reduced penetrance and phenocopies. Most of our calculations have been carried out under the assumption that 50% of families are linked to a marker locus. We have varied both the number of offspring per family and the sampling scheme. We have also investigated the increased power when the disease locus is midway between two marker loci 10 cM apart. For recessive inheritance, both linkage and heterogeneity can be detected in clinically feasible sample sizes. For dominant inheritance, linkage can be detected but heterogeneity cannot be detected unless larger sibships (four offspring) are sampled or two linked markers are available. As expected, if penetrance is reduced, sampling families with all sibs affected is most efficient. Our results provide a basis for estimating the amount of resources needed to find genes for complex disorders under conditions of heterogeneity.     

Effects of D-serine on bacterial D-amino acid transaminase: accumulation of an intermediate and inactivation of the enzyme

Incubation of pure bacterial D-amino acid transaminase with D-serine or erythro-beta-hydroxy-DL-aspartic acid, which are relatively poor substrates, leads to generation of a new absorbance band at 493 nm that is probably the quinonoid intermediate. The 420-nm absorbance band (due to the pyridoxal phosphate coenzyme) decreases, and the 338-nm absorbance band (due to the pyridoxamine phosphate or some other form of the coenzyme) increases. A negative Cotton effect at 493 nm in the circular dichroism spectra is also generated. Closely related D amino acids do not lead to generation of this new absorption band, which has a half-life of the order of several hours. Treatment of the enzyme with the good substrate D-alanine leads to a small but detectable amount of the same absorbance band. D-Serine but not erythro-beta-hydroxyaspartate leads to inactivation of D-amino acid transaminase, and D-alanine affords partial protection. The results indicate that D-serine is a unique type of inhibitor in which the initial steps of the half-reaction of transamination are so slow that a quinonoid intermediate with a 493-nm absorption band accumulates. A derivative formed from this intermediate inactivates the enzyme.     

Behavior of male and female spruce bark beetles,Ips typographus,on the bark of host trees during mass attack

The behavior of 118 spruce bark beetles, Ips typographus,was observed on trees under colonization. Most individuals were followed from when they landed until they entered or left the tree. Both males and females spent most time inspecting crevices and searching for a place to start boring or for a hole to enter. These behaviors accounted for 87 and 70% of all behavioral acts recorded for males and females, respectively. Females entered galleries with males only after a period of pushing at the gallery entrance. Males spent on average 3 min and females 4 min on the bark before entering or leaving the tree. Thirty-three percent of the beetles eventually entered the tree, 31% flew away, 35% dropped from the host, and one beetle was eaten by a predator. The results are discussed in relation to the question of mate choice in bark beetles and to studies on attack dynamics of spruce bark beetle populations.     

Mass cultivation of the nitrogen-fixing cyanobacteriumGloeotrichia natans,indigenous to rice-fields

Gloeotrichia natans, a nitrogen fixing cyanobacterium common in rice fields in the Philippines, was used for studies to establish key features of its physiology and potential production in outdoor cultures. Under optimal growth conditions (38 °C, pH 8.0, no carbon enrichment) the specific growth rate of rice-field isolate was 0.076 h^–1. The pH of the medium (between 6.5 and 9.0) did not influence the growth rate, but it did affect phycobiliprotein content, as reflected by a change in colour. At pH 7.0 the culture was green-brown, with phycobiliproteins constituting up to 10% of the total protein, while at pH 9.0 the culture was brownish-black and the pigment content was as high as 28% of the total protein. In outdoor cultures the specific growth rate was related directly to cell density in the range of 0.7–1.5 g dry weight 1^–1 at a rate of stirring of 30 rpm, and inversely related to cell density at half this rate. At a stirring of 30 rpm, daily production of outdoor cultures harvested to maintain cell densities of 0.7, 1.15 andw 1.5 g 1^–1 were 14.7, 17.1 and 18.1 g m^–2 d^t, respectively. This rate of production was maintained for more than 45 days. Phycobiliprotein content in the culture kept at a density of 1.5 g 1^–1 reached 14% of the total biomass.