Particulate glycogen of mammalian liver: specificity in binding phosphorylase and glycogen synthase.

The glycogen particle - glycogen metabolizing enzyme complex was investigated to gain some understanding of its physiological significance. Fractionations of populations of particles from mouse liver were carried out utilising open column and high performance liquid chromatography, and based either on the molecular weight of the particles or the hydrophobic interactions of the glycogen-associated proteins. The activities of glycogen phosphorylase and glycogen synthase were measured in these fractions. Fractionations were of tissue in different stages of glycogen deposition or mobilization. In animals fed ad libitum, glycogen synthase was associated with the whole spectrum of molecular weights, while the glycogen phosphorylase distribution was skewed in favour of the lower molecular weight species. Under conditions of glycogen mobilization, the phosphorylase distribution changed to include all molecular weights. The hydrophobic interaction separations demonstrated that glycogen synthase binds to a specific subpopulation of particles that is a minor proportion of the total. In general, there was a direct relationship of the total amount of phosphorylase and synthase bound during periods of mobilization and deposition, respectively. Two notable exceptions were the large amounts of glucose-6-P dependent synthase present during the early period of glycogen mobilization and the high amounts of active phosphorylase appearing shortly after food withdrawal, in spite of interim glycogen deposition from presumably already ingested food.

Regulation of glycogen synthesis in human skeletal muscle: does cellular glycogen control glycogen synthase phosphatase activity?

Contrary to the accepted feedback control mechanism of glycogen biosynthesis in skeletal muscle, evidence is presented here leading to the conclusion that glycogen does not control the activity of glycogen synthase phosphatase in intact human skeletal muscle tissue.

Fractionation of particulate glycogen and bound enzymes using high-performance liquid chromatography.

During a study aimed at the specificity of binding of phosphorylase and glycogen synthase to glycogen particles we investigated the separation of these particles with the technique of high-performance liquid chromatography using several media. The technique allowed relatively rapid fractionations with little, if any, loss of enzyme activity. The basis of some separations was molecular weight of the particle. Of the molecular exclusion media used, Superose 6 had an exclusion limit large enough to accommodate most glycogens but excluded mouse liver and bovine liver glycogen particles. Toyopearl HW-75 F was a medium that could accommodate liver glycogen and gave us reasonably well-defined separation of the particles that carried phosphorylase and glycogen synthase. Separations on ion-exchange columns, that were obviously based on the overall charge of proteins associated with the glycogen particles, were not very effective in terms of allowing the separation of different types of particle. Of all the media used the hydrophobic interaction column was best in terms of separating what appeared to be distinct populations of particles that had definite preferences in terms of binding phosphorylase and glycogen synthase. As expected, these separations were based on the hydrophobic interaction of the proteins associated with the glycogen particles, since particles that had been deproteinized by a cold water extraction procedure did not bind to the column.

A novel technique for measuring solute diffusivities in entrapment matrices used in immobilization.

A novel technique has been developed for measuring effective solute diffusivities in entrapment matrices used for cell immobilization. In this technique radiotracers were used to measure effective diffusivities and equilibrium partition coefficients of the solute between the liquid and solid matrix. Ca-alginate was used in this study, because it is one of the most commonly employed matrices for the immobilization of microbial, plant and mammalian cells. The experimental apparatus consisted of a single spherical Ca-alginate bead which was attached to a rotating rod and immersed in water containing C(14)-glucose. The rotational speed of the spherical bead was controlled and resulted in excellent mixing, and negligible external film mass transfer resistance, which allowed the measurement of true effective solute diffusivity within the solid matrix. The rates of C(14)-glucose diffusion within the Ca-alginate sphere were measured using a scintillation spectrometer. A mathematical model of unsteady-state diffusion in a sphere was used with appropriate boundary conditions, and the effective diffusivity of glucose was found from the best fit of the experimental data using a computer regression analysis method. Using 2% (w/v) Ca-alginate beads in this new radiotracer technique the effective diffusivity and partition coefficient of glucose were found to be 6.62 x 10(-10) m(2)/s and 0.98, respectively. The accuracy, advantages, and simplicity of this new method for diffusivity measurements are also compared to other existing methods.

An unusual cyclic AMP-dependent protein kinase from nematodes.

The search for an unusual cyclic nucleotide-dependent protein kinase in nematodes represented an attempt to gain some insight into the proposed homology of the cAMP and cGMP-dependent protein kinases. Two species of protein kinase were found in high speed supernatants of the mycophagous nematode Aphelenchus avenae. One of the two, bound to DEAE cellulose and was eluted from it in a manner characteristic of the type I cAMP kinase. The enzyme had high affinity for cAMP and dissociated upon binding to the cyclic nucleotide, as judged by the fact that catalytic activity did not bind to a cAMP affinity column. The second enzyme did not bind to DEAE. Unexpectedly, it too had high affinity for cAMP and much lower affinity for cGMP (unlike the cAMP/cGMP kinase from insects). The holoenzyme bound tightly to the cAMP affinity column and required a high concentration of the cyclic nucleotide for elution. This latter enzyme is the only example of a cAMP-dependent protein kinase that does not dissociate upon activation.

Pullulan Elaboration by Aureobasidium pullulans Protoplasts.

Protoplasts of Aureobasidium pullulans are capable of producing pullulan. Biosynthesis of the polymer pullulan required induction with kinetics similar to those of whole cells. The protoplasts also produced a heteropolysaccharide component containing mannose, glucose, and galactose. The relative proportions of the pullulan and heteropolysaccharide fractions were a function of glucose concentration, with the pullulan content of the total polysaccharide rising from 20% at 2.5 mM glucose to 45% at 20 mM glucose. Elaboration of pullulan by both cells and protoplasts was sensitive to 0.6 M KCl, which was present as the osmotic stabilizer in protoplast experiments. The presence of KCl resulted in a shift in the pH optimum to a more acidic value. The molecular weight of the protoplast-derived pullulan was sharply reduced from the molecular weight of the whole-cell-derived product. Exposure of the protoplasts to proteolytic enzymes had no effect on polysaccharide elaboration.

Simplified microassay for pullulan synthesis.

A simple radiometric microassay for extracellular polysaccharide elaboration by yeast-like cells of Aureobasidium pullulans was developed, based upon a procedure originally described by Catley (FEBS Lett. 20:174-176, 1972). Incorporation of [C]glucose into pullulan was linear with respect to time and cell dose. The pH and temperature optima for elaboration were 5.3 and 30 degrees C, respectively. Polysaccharide elaboration declined linearly with culture age.

A unique cyclic nucleotide-dependent protein kinase.

During the course of studying the soluble cyclic nucleotide-dependent protein kinases of a developing insect, three different enzymes were isolated. Two of these were found to be cAMP-dependent enzymes eluting from DEAE-cellulose in a manner identical with protein kinases I and II found in vertebrate muscle. The third enzyme appears to be unique. It has high affinity for either cAMP or cGMP (KA of 43 nM and 25 nM, respectively), the only cyclic nucleotide-dependent kinase described, to have this property. The enzyme has lower affinity for cIMP and cCMP (KA of 160 nM and 340 nM, respectively). Binding to cyclic nucleotide does not alter enzyme size. The KM for ATP is 86 microM, and among several types of histones tried, the slightly lysine-rich subgroup f2a was the best phosphate acceptor. Maximum activity was obtained with 1 mM Mg2+ while Mn2+ was completely ineffective. This new enzyme was purified to homogeneity on a cAMP affinity column as judged by two-dimensional electrophoresis. On the basis of molecular sieving and sodium dodecyl sulfate electrophoresis we have reached the preliminary conclusion that the native enzyme is a dimer of identical subunits with a molecular weight of 180,000. If the mammalian cAMP and cGMP enzymes are indeed homologous proteins, perhaps we have in this new kinase a species that represents a common ancestral protein.

New Method of Producing Protoplasts of Aureobasidium pullulans.

A rapid and relatively inexpensive method for producing protoplasts of the black yeast Aureobasidium pullulans is described. The procedure involves anaerobic incubation with the lytic preparation Driselase.

Characteristics of the chitin-synthesizing system of insect tissue.

Results obtained from isotopic dilution experiments are consistent with the operation of the chitin pathway as it has been established in fungal preparations. The last enzyme in this pathway, namely chitin synthase, seems to be accessible to substrate from the cell exterior, indicating its presence in the plasma membrane. It can, however, only be saturated (in a manner that partially excludes reaction with a competing substrate) from the cell side of the membrane.