Ronald D. Edstrom

A sensitive method for the assay of hyaluronidase activity.

Abstract A spectrophotometric method for the assay of hyaluronidase activity, based on the binding of a carbocyanine dye (1-ethyl-2-[3-(1-ethyl-naphtho[1,2 d ]thiazolin-2-ylidene)-2-methyl-propenyl]naphtho[1,2d]thiazolium bromide) to undegraded substrate, is described. The binding results in a spectral shift with an absorbance maximum at 640 nm which is proportional to the amount of hyaluronic acid within a prescribed range. The end products of hyaluronidase activity do not cause a shift in the dye spectrum. The method allows the determination of an amount of hyaluronidase equivalent to 0.00005 NF unit.

Direct visualization of phosphorylase-phosphorylase kinase complexes by scanning tunneling and atomic force microscopy

In skeletal muscle the activation of phosphorylase b is catalyzed by phosphorylase kinase. Both enzymes occur in vivo as part of a multienzyme complex. The two enzymes have been imaged by atomic force microscopy and the results compared to those previously found by scanning tunneling microscopy. Scanning tunneling microscopy and atomic force microscopy have been used to view complexes between the activating enzyme phosphorylase kinase and its substrate phosphorylase b. Changes in the size and shape of phosphorylase kinase were observed when it bound phosphorylase b.

A colorimetric method for the determination of mucopolysaccharides and other acidic polymers.

Abstract A colorimetric method for the estimation of small amounts (0.5 to 5 μg) of acidic polysaccharides has been developed using the shift in the visible absorption spectrum of a carbocyanine dye when it is bound to a polyanion. The limitations of the method including the evaluation of interfering substances have been investigated. The method has the advantages of simplicity and rapidity lacking in methods presently used for mucopolysaccharides and other acidic polysaccharides. The absorbance in the region between 600 and 650 mμ was found to be a linear function of the polyanion concentration.

Scanning tunneling microscopy of the enzymes of muscle glycogenolysis

Scanning tunneling microscopy (STM) has been used to examine the structures of the skeletal muscle enzymes phosphorylase and phosphorylase kinase. The interaction of these two proteins represents the last step in the process of signal transduction which results in muscle glycogen being converted into metabolic energy for use in muscle contraction. Phosphorylase b has a molecular weight of 97,000 and the dimer is seen by STM to have dimensions of 11 X 5.7 nm. Phosphorylase b has a tendency to form linear arrays of dimers on the graphite surface used as the support for STM imaging. Phosphorylase kinase is imaged as a butterfly-like object with lateral dimensions of 36 X 27 nm. The molecular thicknesses given by scanning tunneling microscopy for these two non-conducting molecules is significantly less than expected. The height measurement in STM is dependent not only on the surface topology of the object being imaged, but also on the electronic work function of the object compared to that of the graphite surface on which it lies. In addition to the individual proteins, a complex between phosphorylase and phosphorylase kinase has been observed by scanning tunneling microscopy.

Scanning tunneling microscopy and atomic force microscopy visualization of the components of the skeletal muscle glycogenolytic complex

The muscle glycogenolytic complex is responsible for providing access to the reserve carbohydrate energy stores in skeletal muscle during times of vigorous exercise. The complex is a set of enzymes and regulatory factors that are bound to the carbohydrate storage polymer, glycogen. These components provide the ordered synthesis and utilization of that stored form of glucose. Glycogen and the enzyme proteins, phosphorylase and phosphorylase kinase, have been imaged by atomic force microscopy (AFM) or scanning tunneling microscopy (STM). The images of all three generally correlated well with the known features of those molecules, as measured by traditional physicochemical methods. The exception for all three polymers is that the measured height by STM is in error. In each case, the molecules appear to be only about 30% of their true thickness, as measured by height above the graphite surface. It is clear that both AFM and STM will play important roles in biomedical investigation of macromolecular structures...

Direct observations of enzymes and their complexes by scanning tunneling microscopy

Scanning tunneling microscopy (STM) has been used as a method of studying the relationships between the enzymes of muscle glycogenolysis. In skeletal muscles the activation of phosphorylase b is catalyzed by phosphorylase kinase. This interaction is believed to occur in vivo as part of a multienzyme complex. The molecular structures of phosphorylase b and phosphorylase kinase have been visualized by STM.1 Phosphorylase b can be seen in dimeric and tetrameric forms as well as linear and circular aggregates. Individual molecules of phosphorylase kinase image as planar, bilobate structures with a twofold axis of symmetry and a central depression. STM has also been used to visualize complexes between phosphorylase kinase and its substrate, phosphorylase b.

Levels of blood group synthetic enzymes in human colonic carcinoma.

Summary Homogenates of tumorous and adjacent non-tumorous colorectal tissues from 18 patients were tested for the activities of blood group synthetic enzymes, namely α-D-galactosyl transferase for B isoantigen and α-N-acetyl-D-galactosamine transferase of A isoantigen. The galactosyl transferase activity in non-tumorous intestinal tissue was high in patients with blood group B, intermediate in group AB and was absent in group A or O. As compared with adjacent non-tumorous tissue, the Gal transferase activity increased in tumors by 1.6- to 6.9-fold in four of five patients with blood group B or AB, and the α-N-acetyl-D-galactosaminyl transferase activity increased in three- of six-tumor tissues from patients of blood group A or AB. The results suggest that the reported losses of ABH isoantigen in colorectal cancer are not due to deficiencies of Gal or GalNAc transferase activities involved in the synthesis of blood group antigens.

Observation of phosphorylase kinase and phosphorylase b at solid–liquid interfaces by scanning tunneling microscopy

Enzymes can be immobilized at a solid–liquid interface and observed with a scanning tunneling microscope (STM) using the inherent charge of proteins and an external potential applied to the STM substrate. Phosphorylase kinase, and dimers and oligomers of phosphorylase b have been observed at the interface of aqueous solutions and highly oriented pyrolytic graphite (HOPG). The lateral dimensions of phosphorylase kinase determined by STM at the solid–liquid interface are from 74%–78% of the dimensions determined by STM at the solid–air interface [Biochem. 28, 4939 (1989); Biophys. J. (in press)]. The phosphorylase b lateral dimensions of both enzymes are between 1.9 and 2.5 nm greater than the dimensions determined by x‐ray crystallography. The vertical dimensions determined by STM at the solid–liquid and solid–air interfaces are in reasonable agreement with each other. Mixtures of the two enzymes show aggregates in which the complexes of the two enzymes are identifiable. This technique will make it possibl...

Mechanism of spectral changes of a carbocyanine dye with acidic polysaccharides and oligogalacturonides.

Abstract Changes in the visible spectrum of a cationic carboeyanine dye in the presence of α (1–4) linked oligomers of d -galacturonic acid have been found to be dependent on the number of uronic acid residues in the molecule. Polygalacturonic acid caused a shift in the dye spectrum that was linearly proportional to the polymer concentration. Neither mono- nor digalacturonic acid had an effect on the dye spectrum. Tri- and tetragalacturonic acid caused spectral changes which were nonlinear with respect to oligomer concentration while penta- and hexagalacturonic acid showed concentration-dependent properties similar to polygalacturonic acid. The difference spectra with polygalacturonate and other acidic polysaccharides containing one anionic site per monosaccharide residue showed two absorption maxima in the region of 550 nm and 610 nm. All of the oligomers tested (containing 3 through 6 galacturonic acid residues) yielded only a single maxima for each in the region between 650 and 670 nm. This single maxima phenomenon was also observed with acidic polysaccharides having only one anionic site for every two monosaccharide residues (hyaluronic acid and chondroitin).

Mechanism of calmodulin inhibition of cAMP-Dependent protein kinase activation of phosphorylation kinase

The activation of phosphorylase kinase (EC 2.7.1.38; ATP:phosphorylase b phosphotransferase) by the catalytic subunit of cAMP-dependent protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) is inhibited by calmodulin. The mechanism of that inhibition has been studied by kinetic measurements of the interactions of the three proteins. The binding constant for calmodulin with phosphorylase kinase was found to be 90 nM when measured by fluorescence polarization spectroscopy. Glycerol gradient centrifugation studies indicated that 1 mol of calmodulin was bound to each phosphorylase kinase. Phosphorylation of the phosphorylase kinase did not reduce the amount of calmodulin bound. Kinetic studies of the activity of the catalytic subunit of cAMP-dependent protein kinase on phosphorylase kinase as a function of phosphorylase kinase and calmodulin concentrations were performed. The results of those studies were compared with mathematical models of four different modes of inhibition: competitive, noncompetitive, substrate depletion, and inhibition by a complex between phosphorylase kinase and calmodulin. The data conform best to the model in which the inhibitory species is a complex of phosphorylase kinase and calmodulin. The complex apparently competes with the substrate, phosphorylase kinase, which does not have exogenous calmodulin bound to it. In contrast, the phosphorylation of the synthetic phosphate acceptor peptide, Kemptide, is not inhibited by calmodulin.