Two-dimensional electron microscopic analysis of the chalice form of phosphorylase kinase

Phosphorylase kinase (Mr 1.3 X 10(6], a Ca2+-calmodulin-dependent protein kinase, plays a key role in the initiation of glycogenolysis. After purification on hydroxylapatite, the negatively stained enzyme was used for electron microscopy. In electron micrographs, phosphorylase kinase shows two major molecular forms: a butterfly form (approx. 60%) and a chalice form (approx. 40%). Images of the chalice form of the enzyme were computer-averaged by the method of single particle averaging. The following apparent molecular dimensions were obtained from the averages: total height, 20 nm; maximal width, 18 nm. The chalice form of phosphorylase kinase consists of a major structure termed the cup (11 nm X 18 nm), containing a large accessible cleft, and a minor structure termed the stem (8 nm X 9 nm). A closer examination of the images by averaging of molecular parts revealed two subpopulations of the cup part: a flexed (closed) type and an extended (open) type. The orifice, which can be closed partly by two protrusions (I, I'), is about 6 nm wide when the protrusions are flexed and 9 nm wide when they are extended. It is suggested that the substrates, e.g. phosphorylase b, may be accommodated in the large cleft of the enzyme. While the orientation of the protrusions (I, I') is the most obvious difference between the two types, more structural differences can be detected, suggesting a concerted movement of the protein domains against each other.     

Interaction sites on phosphorylase kinase for calmodulin

Holophosphorylase kinase was digested with Glu-C specific protease; from the peptide mixture calmodulin binding peptides were isolated by affinity chromatography and identified by N-terminal sequence analysis. Two peptides originating from the subunit, having a high tendency to form a positively charged amphiphilic helix and containing tryptophane, were synthesized. Additionally, a homologous region of the subunit and a peptide from the subunit present in a region deleted in the isoform were also selected for synthesis. Binding stoichiometry and affinity were determined by following the enhancement in tryptophane fluorescence occurring upon 1:1 complex formation between these peptides and calmodulin. Finally, Ca²⁺ binding to calmodulin in presence of peptides was measured. By this way, the peptides 542–566, 547–571, 660–677 and 597–614 have been found to bind specifically to calmodulin.Together with previously predicted and synthesized calmodulin binding peptides four calmodulin binding regions have been characterized on each the and subunits. It can be concluded that endogenous calmodulin can bind to two calmodulin binding regions in as well as to two regions in and . Exogenous calmodulin can bind to two regions in and in . A binding stoichiometry of 0.8mol of calmodulin/ protomer of phosphorylase kinase has been determined by inhibiting the ubiquitination of calmodulin with phosphorylase kinase. Phosphorylase kinase is half maximally activated by 23nM calmodulin which is in the affinity range of calmodulin binding peptides from to calmodulin. Therefore, binding of exogenous calmodulin to activates the enzyme. A model for switching endogenous calmodulin between , and and modulation of ATP binding to as well as Mg²⁺/ADP binding to by calmodulin is presented.     

A novel nanolayer biosensor principle

A method for eliminating the mass transport limitation on biosensor surfaces is introduced. The measurement of macromolecular binding kinetics on plane surfaces is the key objective of many evanescent wave (e.g. total internal reflection fluorescence (TIRF)), and surface plasmon resonance (SPR) based biosensor systems, allowing the determination of binding constants within minutes or hours. However, these methods are limited in not being rigorously applicable to large macromolecules like proteins or DNA, since the on-rates are transport limited due to a Nernst diffusion layer of 5-10 microm thickness. Thus, for the binding of fibrinogen (340 kDa) to a surface current SPR biosensors will show a mass transport coefficient of ca. 2 x 10(-6) m/s. In a novel approach with an immiscible fluid vesicle (e.g. air bubble), it has been possible to generate nanoscopic fluid films of ca. 200 nm thickness on the sensor surface of an interfacial TIRF rheometer system. The thickness of the liquid film can be can be easily probed and measured by evanescent wave technology. This nanofilm technique increases the mass transport coefficient for fibrinogen to ca. 1 x 10(-4) m/s eliminating the mass transport limitation, making the binding rates reaction-rate limited. From the resulting exponential kinetic functions, lasting only 20-30s, the kinetic constants for the binding reaction can easily be extracted and the binding constants calculated. As a possible mechanism for the air bubble effect it is suggested that the aqueous fluid flow in the rheometer cell is separated by the air bubble below the level of the Nernst boundary layer into two independent laminar fluid flows of differing velocity: (i) a slow to stationary nanostream ca. 200 nm thick strongly adhering to the surface; and (ii) the bulk fluid streaming over it at a much higher rate in the wake of the air bubble. Surprising properties of the nanofluidic film are: (i) its long persistence for at least 30-60s after the air bubble has passed (2.5s); and (ii) the absence of solute depletion. It is suggested that a new liquid-liquid interface (i.e. a "vortex sheet") between the two fluid flows plays a decisive role, lending metastability to the nanofluidic film and replenishing its protein concentration via the vortices-thus upholding exponential binding kinetics. Finally, the system relaxes via turbulent reattachment of the two fluid flows to the original velocity profile. It is concluded that this technique opens a fundamentally novel approach to the construction of macromolecular biosensors.     

Characterization of PR-10 genes from eight Betula species and detection of Bet v 1 isoforms in birch pollen

Background  

Bet v 1 is an important cause of hay fever in northern Europe. Bet v 1 isoforms from the European white birch (Betula pendula) have been investigated extensively, but the allergenic potency of other birch species is unknown. The presence of Bet v 1 and closely related PR-10 genes in the genome was established by amplification and sequencing of alleles from eight birch species that represent the four subgenera within the genus Betula. Q-TOF LC-MS^E was applied to identify which PR-10/Bet v 1 genes are actually expressed in pollen and to determine the relative abundances of individual isoforms in the pollen proteome.     

ATP-dependent proteolysis and the role of ubiquitin in rabbit cardiac muscle

A soluble ATP/Mg2-dependent proteolytic system from rabbit cardiac muscle has been identified (m ca. 310 kDa) and purified ca. 9-fold. This enzyme which splits the substrate [3H]globin and 125I-bovine serum albumin (125I-BSA) has many similarities to the ATP-dependent proteolytic enzyme system from reticulocytes which utilizes ubiquitin: 1) The specific activities in reticulocyte lysates and cardiac muscle extracts are of the same magnitude (0.5-1 arb. unit/mg). 2) The binding and elution behavior on DEAE-cellulose is similar. 3) In both cases the pH optimum (substrate 125I-BSA) is pH 7.6. 4) Both enzymes are inhibited by hemin, NEM and iodoacetate but not e.g. by leupeptin, or inhibitors of serine proteases. 5) Neither enzyme system can utilize ATP-analogs such as AMP-CPP, AMP-PCP, AMP-PNP or ATP-gamma-S. There are however also significant differences: 1) The enzyme system from cardiac muscle is fully active in the absence of ubiquitin and cannot be activated by this peptide. 2) The enzyme from cardiac muscle can degrade methylated BSA. 3) The cardiac muscle enzyme can be further purified on Sepharose 4B; the enzyme from reticulocytes is inactivated by this procedure. 4) The cardiac enzyme cannot be inactivated by ribonuclease as the reticulocyte counterpart. Although ubiquitin does not appear to play a role in the isolated ATP/Mg2-dependent proteolytic system from cardiac muscle, it is demonstrated for the first time that 125I-ubiquitin can be conjugated to a wide variety of cardiac muscle proteins in vitro in an ATP-dependent manner. Apparent molecular masses of major conjugates were: 185 kDa, 140 kDa, 85 kDa, 65 kDa, 46 kDa, 38 kDa and 36 kDa as estimated by discontinuous SDS gel electrophoresis. Addition of purified phosphorylase kinase to cardiac muscle extract changed the ubiquitination pattern by the appearance of two novel protein bands. It is concluded that the ATP/Mg2-dependent proteolytic system of cardiac muscle must be differentiated from the proteolytic system of reticulocytes mainly because of its ubiquitin-independence. Nevertheless the conjugation of 125I-ubiquitin to many muscle proteins is a strong indication for a crucial role of this interesting peptide in striated muscle.     

Recent advances in ABE fermentation: hyper-butanol producing Clostridium beijerinckii BA101

This is an overview of the mutant strain Clostridium beijerinckii BA101 which produces solvents (acetone–butanol–ethanol, ABE) at elevated levels. This organism expresses high levels of amylases when grown on starch. C. beijerinckii BA101 hydrolyzes starch effectively and produces solvent in the concentration range of 27–29 g l⁻¹. C. beijerinckii BA101 has been characterized for both substrate and butanol inhibition. Supplementing the fermentation medium (MP2) with sodium acetate enhances solvent production to 33 g l⁻¹. The results of studies utilizing commercial fermentation medium and pilot plant-scale reactors are consistent with the results using small-scale reactors. Pervaporation, a technique to recover solvents, has been applied to fed-batch reactors containing C. beijerinckii BA101, and solvent production as high as 165 g l⁻¹ has been achieved. Immobilization of C. beijerinckii BA101 by adsorption and use in a continuous reactor resulted in reactor productivity of 15.8 g l⁻¹ h⁻¹. Recent economic studies employing C. beijerinckii BA101 suggested that butanol can be produced at US$0.20–0.25 lb⁻¹ by employing batch fermentation and distillative recovery. Application of new technologies such as pervaporation, fed-batch culture, and immobilized cell reactors is expected to further reduce these prices. Journal of Industrial Microbiology & Biotechnology (2001) 27, 287–291. Received 12 September 2000/ Accepted in revised form 27 January 2001     

General aspects of hydrophobic chromatography. Adsorption and elution characteristics of some skeletal muscle enzymes.

If the degree of substitution of Sepharose 4 B with alpha-alkylamines is varied gels of different hydrophobicity are produced. Proteins can be adsorbed when a critical hydrophobicity (ca. 10-12 alkyl residues/Sepharose sphere) is reached. The enzymes phosphorylase kinase, phosphorylase phosphatase, 3',5'-cAMP dependent protein kinase, glycogen synthetase, and phosphorylase b are successively adsorbed as the hydrophobicity of the Sepharose is increased. The capacity of the gels for these enzymes and protein in general increases exponentially reaches plateau values as a function of the degree of substitution. There is no indication of a restriction of the hydrophobic centers for a given protein. The critical hydrophobicity needed to adsorb proteins can either be otained in the above manner or by elongation of the employed alkylamine at a constant degree of substitution. Additonally, as the hydrophobicity of a gel is increased higher binding forces result and desorption of proteins requires an augmentation of the salt concentration in the elution buffer. Elution of proteins from a hydrophobic matrix can be described in terms of salting-in phenomena since desorption is dependent on the type of salt employed and not on the ionic strength alone. This also rules out ionic interactions as a major factor in adsorption per se. By rationally controlling the hydrophobicity of a Sepharose gel the adsorption and elution of a protein may be thus establised that its purification or elimination can be optimally performed.