Purification and Characterization of an Extracellular Alkaline Phosphatase from Penicillium Chrysogenum

Abstract

An extracellular alkaline phosphatase from Penidllium chrysogenum was purified to homogeneity using DEAE ion-exchange chromatography and size exclusion chromatography. SDS-PAGE of the purified enzyme indicated a molecular weight of 58,000. The mobility of the native enzyme on a Superose 12 column suggests that the active form of the enzyme is a monomer. The enzyme catalyzes the hydrolysis of phosphate from a variety of substrates including p-Miitrophenyl phosphate, α-naphthyl phosphate and the anti-tumor compound etoposide phosphate. The apparent K_m for the substrate p-nitrophenyl phosphate is 1.3 mM and the enzyme is inhibited by inorganic phosphate. The pH optimum of the enzyme is 9.0 with a broad optimal temperature range between 40 and 50 °C. The isoelectric point of the enzyme is approximately 5.5. The enzyme is a glycoprotein; digestion with endoglycosidase H indicates that the protein consists primarily of N-inked carbohydrates. Enzymatic activity is enhanced by the addition of divalent cations such as Mg⁺⁺ and Mn⁺⁺ and inhibited by addition of a chelator such as EDTA suggesting a metal ion requirement. The enzyme was found to be an inexpensive catalyst for the conversion of etoposide phosphate to etoposide in the manufacture of this anti-tumor compound.     

Extending a biologically inspired model of choice: multi-alternatives, nonlinearity and value-based multidimensional choice

The leaky competing accumulator (LCA) is a biologically inspired model of choice. It describes the processes of leaky accumulation and competition observed in neuronal populations during choice tasks and it accounts for reaction time distributions observed in psychophysical experiments. This paper discusses recent analyses and extensions of the LCA model. First, it reviews the dynamics and examines the conditions that make the model achieve optimal performance. Second, it shows that nonlinearities of the type present in biological neurons improve performance when the number of choice alternatives increases. Third, the model is extended to value-based choice, where it is shown that nonlinearities in the value function explain risk aversion in risky choice and preference reversals in choice between alternatives characterized across multiple dimensions.     

Laboratory studies of predation by the Antarctic mite Gamasellus racovitzai (Acarina: Mesostigmata)

Summary Laboratory investigations of predation by Gamasellus racovitzai (Acarina: Mesostigmata) on Cryptopygus antarcticus (Insecta: Collembola) are described. The predator appeared to search at random, but, when contact with prey had been made, a rapid attack involved looping the forelegs over the prey to hold it whilst the chelicerae moved forward horizontally to puncture the side of the prey. The mean predation rate by deutonymphs, approximately one prey per predator per 12 days, was independent of prey density, but with adults this rate increased to about one prey per predator per 3 days. A study of leg geometry predicted a maximum prey size that could be captured: some experimental evidence suggested that mites selected prey near to this predicted size. The contribution of the laboratory results towards understanding the dynamics of field populations is discussed.     

Allotypes associated with B apolipoproteins in rabbits

Western blot analysis of the alloantisera (i.e., anti-Lpq1, anti-Lpq2, anti-Lpq3, and anti-Lpq4) which defined the three lpq genes of rabbit linkage group VIII showed that they reacted strongly with an apolipoprotein of molecular weight 320,000. They also cross-reacted with an apolipoprotein of molecular weight 220,000. The two apolipoproteins that reacted with the alloantisera were found by SDS-polyacrylamide gel electrophoresis to be present in very low density (VLDL), intermediate density (IDL), and low density (LDL) lipoprotein fractions and by Western blot analysis to react with an anti-apolipoprotein B antiserum. These results support the conclusion that the alloantisera react with allotypes associated with the B apolipoproteins. The distribution of the four allotypes among different lipoprotein fractions, however, differed. The quantitative competitive Enzyme Linked Immunosorbant Assay (ELISA) showed that the Lpq1, Lpq2, and Lpq4 allotypes were found in the highest concentration in VLDL, IDL, and LDL, and in significantly lower concentrations in plasma chylomicrons. The concentrations of these allotypes in high density lipoproteins (HDL) as measured in the ELISA were about 1% of the concentrations found in LDL. The Lpq3 allotype, on the other hand, was present in the highest concentrations only in IDL and LDL and in significantly lower concentrations in VLDL and plasma chylomicrons. Surprisingly, the concentration of the Lpq3 allotype in HDL was 20% of the level found in LDL.     

Synthesis of 2'(3')-O-DL-alanyl hexainosinic acid using T4 RNA ligase: suppression of the enzymic reverse transfer reaction by alkaline phosphatase.

2'(3')-O-DL-Alanyl (Ip)5I was synthesized by a new method. An alanine ortho ester of inosine 5'-phosphate was added to (Ip)4I using the ATP-independent reaction of T4 RNA ligase, and the product was converted smoothly to the desired ester. The enzymic reverse transfer reaction was conveniently suppressed by the dephosphorylation of the adenosine 5'-phosphate coproduct, catalyzed in situ by alkaline phosphatase.     

Human adipsin is identical to complement factor D and is expressed at high levels in adipose tissue.

A cDNA for human adipsin was isolated and shown to encode a protein sharing 98% amino acid sequence similarity with the protein sequence previously determined for purified natural human complement factor D. Like mouse adipsin, recombinant human adipsin displays the enzymatic activity of human complement factor D, cleaving complement factor B only when B is complexed with activated complement component C3. We conclude that human adipsin is equivalent to complement factor D and that adipsin is the homologue of factor D in rodents. Adipose tissue is a major site of synthesis of human adipsin/complement factor D mRNA, but unlike the case in rodents, human adipsin mRNA is also expressed in monocytes/macrophages. The data presented here, demonstrating the equivalence of human adipsin to complement factor D and its high level of expression in fat, suggest a previously unsuspected role for adipose tissue in immune system biology.     

Two spiders in subfamily Mynogleninae (Araneae: Linyphiidae) from the Falkland Islands, South Atlantic

Two new taxa, Falklandoglenes spinosa gen. nov. and sp. nov. and Beauchenia striata gen. nov. and sp. nov. , are described from Beauchêne Island, the most remote island in the Falkland Islands archipelago. Both species are in subfamily Mynogleninae, previously thought to be confined to Central Africa and to New Zealand and its neighbouring subantarctic islands. Both species show intermediate characters between the Mynogleninae and the remainder of the Linyphiidae, and hence they throw some light on the phylogeny of this large, complex, world–wide family of spiders.