The primary structure of the a and (3 subunits ofphosphorylase kinase reveals that both proteins contain a carboxyl-terminal CA1A2X motif (where C is cysteine, Al and A2 are aliphatic amino acids, and X is an uncharged amino acid), the recognition signal for a protein polyisoprenyltransferase. The product, a polyisoprenylated cysteine, can be detected by phenylthiocarbamoylamino acid analysis and by microsequencing following conversion to S-ethylcysteine. Mass spectrometry confirms a covalently linked farnesyl residue in both subunits. Tandem mass spectrometry localizes these modifications at the cysteine residues present in the carboxylterminal CAMQ and CLVS sequences of the a and 3 subunits, respectively. Membrane association of phosphorylase kinase, probably mediated by these farnesyl residues, is discussed. be dictated by the amino acid X. If X represents methionine or serine, the motifis recognized by farnesyltransferases (24), whereas a carboxyl-terminal leucine or phenylalanine specifies geranylgeranylation (25,26 Phosphorylase kinase is found in two distinct compartments in muscle cells. The bulk of the enzyme is present in the cytosol, associated preferentially with glycogen particles. There, it can trigger a rapid conversion of glycogen phosphorylase b to the active a form, switching on glycogenolysis to meet energy demands of working muscle (1). A small fraction of phosphorylase kinase, however, is membraneassociated (2-4). Monoclonal or polyclonal monospecific antibodies localize all four subunits (a, A, y, and 8) of phosphorylase kinase at the sarcoplasmic reticulum, especially at the surface of terminal cisternae facing the T-tubule (5, 6).Phosphorylase kinase can enhance the activity of the sarcoplasmic reticulum Ca2+-transport ATPase (7) due to generation of phosphatidylinositol 4-phosphate, a lipophilic effector of this ATPase (8). Indeed, purified phosphorylase kinase catalyzes phosphorylation of phosphatidylinositol; attempts to separate this phosphatidylinositol 4-kinase from phosphorylase kinase were not successful (9).The carboxyl terminus of both the a and (3 subunits of phosphorylase kinase contains a CA1A2X motif (10,11), where C is cysteine, Al and A2 are aliphatic residues, and X is an uncharged amino acid. In this motif cysteine can be polyisoprenylated (12,13). Upon thioether formation, further posttranslational modifications may occur, such as removal ofthe three terminal amino acids and subsequent methylation of the newly formed terminal carboxyl group (14-17). Polyisoprenylation was detected first in fungal and yeast mating factors (18,19) and subsequently in regulatory proteins of eukaryotic cells (14, 20-22). In animal cells only three types of proteins are known to be farnesylated and only four to be geranylgeranylated, even though many more proteins contain a carboxyl-terminal CAjA2X motif (reviewed in ref. 23). The specificity of the protein polyisoprenyltransferase appears to MATERIALS AND METHODS Endoproteinase Lys-C was from Boehringer Mannheim, ethanethiol from Ja...

In most applications of biotechnology and downstream processing proteins are exposed to fluid stresses in various flow configurations which often lead to the formation of unwanted protein aggregates. In this paper we present physical degradation experiments for proteins under well-defined flow conditions in a four-roll apparatus. The flow field was characterized numerically by computational fluid dynamics (CFD) and experimentally by particle image velocimetry (PIV). The local shear strain rate as well as the local shear and elongation rate was used to characterize the hydrodynamic stress environment acting on the proteins. Lysozyme was used as a model protein and subjected to well-defined fluid stresses in high and low stress environment. By using in situ turbidity measurements during stressing the aggregate formation was monitored directly in the fluid flow. An increase in absorbance at 350 nm was attributed to a higher content of visible particles (>1 µm). In addition to lysozyme, the formation of aggregates was confirmed for two larger proteins (bovine serum albumin and alcohol dehydrogenase). Thus, the presented experimental setup is a helpful tool to monitor flow-induced protein aggregation with high reproducibility. For instance, screening experiments for formulation development of biopharmaceuticals for fill and finish operations can be performed in the lab-scale in a short time-period if the stress distributions in the application are transferred and applied in the four-roll mill.

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