Edmond H. Fischer

The phosphorylase b to a converting enzyme of rabbit skeletal muscle

Abstract A method for the purification of the enzyme catalyzing the conversion of phosphorylase b to a is described. After a 65-fold increase in specific activity, the enzyme obtained is free of PR enzyme activity. The course of the reaction at several concentrations of converting enzyme is illustrated, and converting enzyme units are defined. The optimum pH for the enzyme is approximately 9.0; the reaction requires Mn ++ or Mg ++ ions and ATP. It is shown that a mono-manganous-ATP complex is probably acting in the reaction. Conversion of phosphorylase b to a is carried out in the presence of 32 P-ATP, and an incorporation of at least 2 moles of 32 P per mole of phosphorylase a is found to occur.

[49a] Muscle phosphorylase b: x Glucose-1-phosphate+ Gn ⇄ Gn+x + x inorganic phosphate(where Gn designates glycogen containing n glucose residues)

Publisher Summary This chapter describes an assay method, purification procedure, and properties of muscle phosphorylase b . The assay is carried out as described in the presence of adenosine monophosphate (AMP), since phosphorylase b is enzymatically inactive in the absence of this nucleotide. Phosphorylase b is crystallized by the addition of AMP and magnesium acetate to a partially purified ammonium sulfate fraction of muscle extract. The extraction procedure is designed to give large yields of phosphorylase and to make certain that all this enzyme is in the form of phosphorylase b . These extracts are not suitable for conversion of the enzyme to the a form at the crude extract stage, but the isolated phosphorylase b can be readily converted to phosphorylase a with purified phosphorylase b kinase. Phosphorylase b is enzymatically inactive in the absence of AMP. Crystalline phosphorylase b is homogeneous in the electrophoresis apparatus and the ultracentrifuge. Phospborylase b is much more soluble than phosphorylase a in a glycerophosphate-cysteine buffer, pH 7, and does not crystallize spontaneously like this latter enzyme, even at a concentration of 50 mg/ml at 0°.

Adenosine 5′-0(3-thiotriphosphate) in the control of phosphorytase activity†

Abstract Rabbit muscle phosphorylase b (EC 2.4.1.1) is converted to a thio-analog of phosphorylase a by phosphorylase kinase, Mg2+ and adenosine 5′-O(3-thiotriphosphate)(ATPγS). Conversion proceeds at one-fifth the rate obtained with ATP though the extent of reaction and final level of activation of the enzyme are the same. However, the thiophosphorylase a produced is resistant to phosphorylase phosphatase and, therefore, behaves as a competitive inhibitor with a KI of 3 μM, similar to the KM obtained with normal phosphorylase a . ATPγS can also be utilized by protein kinase in the activation of phosphorylase kinase at a rate similar to that obtained with ATP. It is hydrolyzed at 5 to 10 times the normal rate by the sarcoplasmic reticulum ATPase. When added to a muscle glycogen-particulate complex in the presence of Ca2+ and Mg2+, ATPγS triggers an activation of phosphorylase with simultaneous inhibition of phosphorylase phosphatase as previously observed with ATP.

CD45 regulates signal transduction and lymphocyte activation by specific association with receptor molecules on T or B cells

Evidence is presented that the leukocyte common antigen CD45 can regulate both signal transduction by lymphocyte receptor molecules and T- and B-cell proliferation in a manner dependent on specific interactions between these receptors on the cell surface. Formation of homoaggregates of CD3, CD2, or CD28 on the surface of T cells induced by crosslinking with monoclonal antibodies (mAbs) results in an increase in cytoplasmic free calcium concentration ([Ca2+]i). This increase in [Ca2+]i was abolished when these receptors were crosslinked to CD45 on the cell surface. In contrast, the increase in [Ca2+]i induced by formation of homoaggregates of CD4 was strongly amplified when CD4 was coupled to CD45. T-cell proliferation initiated by immobilized anti-CD3 was inhibited by anti-CD45 or anti-CD45R when immobilized on the same surface, but not when in solution. Similarly, proliferation after stimulation of the CD2 and CD28 receptors was inhibited when a CD45 mAb was crosslinked to either CD2 or CD28 mAbs, but not when a CD45-specific mAb was bound to the cell surface separately. In B cells, the increase in [Ca2+]i and resulting proliferation induced by crosslinking either the CD19 or Bgp95 receptors was inhibited by coupling these molecules to CD45. Thus, CD45 appears to modify other cellular receptors functionally when brought into close physical association with them. The homology of the CD45 conserved cytoplasmic domains with a major human placental protein tyrosine phosphatase suggests that the effects of CD45 described here result from alterations in the phosphorylation state of tyrosyl residues in membrane-associated proteins.

Effect of microinjection of a low-Mr human placenta protein tyrosine phosphatase on induction of meiotic cell division in Xenopus oocytes.

Homogeneous preparations of a protein phosphatase that is specific for phosphotyrosyl residues (protein tyrosine phosphatase [PTPase] 1B) were isolated from human placenta and microinjected into Xenopus oocytes. This resulted in an increase in activity of up to 10-fold over control levels, as measured in homogenates with use of an artificial substrate (reduced carboxamidomethylated and maleylated lysozyme). Microinjected PTPase was stable for at least 18 h. It is distributed within the oocyte in a manner similar to the endogenous activity and is suggestive of an interaction with cellular structures or molecules located predominantly in the animal hemisphere. The phosphatase markedly retarded (by up to 5 h) maturation induced by insulin. This, in conjunction with the demonstration that PTPase 1B abolished insulin stimulation of an S6 peptide (RRLSSLRA) kinase concomitant with a decrease in the phosphorylation of tyrosyl residues in a protein with the same apparent Mr as the beta subunit of the insulin and insulinlike growth factor 1 receptors (M. F. Cicirelli, N. K. Tonks, C. D. Diltz, E. H. Fischer, and E. G. Krebs, submitted for publication), provides further support for an essential role of protein tyrosine phosphorylation in insulin action. Furthermore, maturation was significantly retarded even when the PTPase was injected 2 to 4 h after exposure of the cells to insulin. PTPase 1B also retarded maturation induced by progesterone and maturation-promoting factor, which presumably do not act through the insulin receptor. These data point to a second site of action of the PTPase in the pathway of meiotic cell division, downstream of the insulin receptor and following the appearance of active maturation-promoting factor.

8 Phosphoprotein Phosphatases

Publisher Summary This chapter presents the classification and properties of cytoplasmic phosphoseryl-, phosphothreonyl-, as well as the phosphotyrosyl-protein phosphatases. A breakthrough in the area of phosphatase categorization occurred when Lee et al. found that treatment of the high-molecular-weight enzyme with 80% ethanol converted it to a low-molecular-weight form of Mr∼35,000. This, along with metal ion sensitivity, formed the basis of some early attempts to subdivide the enzymes. However, it soon became apparent that the Mr∼35,000 phosphatase preparations contained two distinct enzymes that could be distinguished by their susceptibilities to the heat-stable inhibitors, as well as their relative activities toward the α- and β-subunits of phosphorylase kinase. These properties have provided the grounds for a new classification system for the phosphoseryl- and phosphothreonyl-protein phosphatases. Studies suggested that the level of phosphotyrosyl residues in cells would reflect the relative activities of the competing kinases and phosphatases in which the type 1 enzymes are those that are susceptible to inhibitor- 1 and -2 and preferentially dephosphorylate the β-subunit of phosphorylase kinase, whereas the type 2 enzymes are not inhibited and display higher activity toward the α-subunit. Types 1 and 2A seem to be the most closely related; they have broad substrate specificities and attack essentially the same phosphate groups but at different rates. Phosphatase-1 has a specific activity toward phosphorylase a approximately 10-fold higher than that of type 2A. Phosphatase-2B (calcineurin) is clearly different because of its stimulation by Ca2+-CaM. The type 2C enyzme shows an absolute dependence on divalent metal ions, such as Mg2+; it acts poorly on phosphorylase a but is very active toward hydroxymethylglutaryl (HMG)-CoA reductase and HMG CoA reductase kinase.

The pure inhibitor of cAMP-dependent protein kinase initiates Xenopus laevis meiotic maturation: A 4-step scheme for meiotic maturation

The availability of the pure inhibitor of cAMP-dependent protein kinase prompted a re-examination of the inhibitor-induced meiotic maturation of Xenopus laevis oocytes. Injection of the inhibitor (1.5 microM) triggered 100% germinal vesicle breakdown faster than progesterone and slower than the maturation-promoting factor: at 0.15 microM, the inhibitor still triggered 100% meiosis, but with a much slower kinetics. In contrast, injection of 24 microM calmodulin resulted in less than 50% GVBD, and results were variable from female to female. Combined injection of inhibitor and calmodulin failed to show any synergism, which does not favour hypotheses according to which calmodulin acts by activation of cyclic nucleotide phosphodiesterase. The net effect of the inhibitor is to decrease the concentration of the free catalytic sub-unit of cAMP-dependent protein kinase, fully dissociated in the unstimulated oocyte, as shown by the absence of effect of pretreatment with cholera toxin on the inhibitor-induced maturation. After such decrease by about 1 microM, a maturation protein, Mp-P, is dephosphorylated by phosphoprotein phosphatases. Dephospho-Mp triggers the synthesis of MPF in cycloheximide-sensitive steps. Finally, MPF triggers GVBD in steps insensitive to cycloheximide. Evidence for such a 4-step scheme--fall in cAMP levels, then in C sub-unit levels, dephosphorylation of Mp leading to the synthesis of MPF and finally MPF-triggered GVBD--is presented and discussed.

Cloning and Characterization of Rat Density-Enhanced Phosphatase-1, a Protein Tyrosine Phosphatase Expressed by Vascular Cells

We have cloned from cultured vascular smooth muscle cells a protein tyrosine phosphatase, rat density-enhanced phosphatase-1 (rDEP-1), which is a probable rat homologue of DEP-1/HPTP eta. rDEP-1 is encoded by an 8.7-kb transcript and is expressed as a 180- to 220-kD protein. The rDEP-1 gene is located on human chromosome 11 (region p11.2) and on mouse chromosome 2 (region 2E). The cDNA sequence predicts a transmembrane protein consisting of a single phosphatase catalytic domain in the intracellular region, a single transmembrane domain, and eight fibronectin type III repeats in the extracellular region (GenBank accession number U40790). In situ hybridization analysis demonstrates that rDEP-1 is widely expressed in vivo but that expression is highest in cells that form epithelioid monolayers. In cultured cells with epitheliod morphology, including endothelial cells and newborn smooth muscle cells, but not in fibroblast-like cells, rDEP-1 transcript levels are dramatically upregulated as population density increases. In vivo, quiescent endothelial cells in normal arteries express relatively high levels of rDEP-1. During repair of vascular injury, expression of rDEP-1 is downregulated in migrating and proliferating endothelial cells. In vivo, rDEP-1 transcript levels are present in very high levels in megakaryocytes, and circulating plates have high levels of the rDEP-1 protein. In vitro, initiation of differentiation of the human megakaryoblastic cell line CHRF-288-11 with phorbol 12-myristate 13-acetate leads to a very strong upregulation of rDEP-1 transcripts. The deduced structure and the regulation of expression of rDEP-1 suggest that it may play a role in adhesion and/or signaling events involving cell-cell and cell-matrix contact.

PHOSPHORYLASE AND RELATED ENZYMES OF GLYCOGEN METABOLISM.

Publisher Summary The finding that pyridoxal phosphate is an essential constituent or coenzyme of glycogen phosphorylase created a new dimension for thought concerning the functions of vitamin B6. This chapter explores questions, such as “Did the discovery of vitamin B6 in phosphorylase stimulate avenues of approach that have led to new disclosures concerning functions of vitamin B6? Have any of the classical findings of vitamin B6 deficiency been explained as a consequence of impaired phosphorylase activity?” The chapter reviews the progress that has been made in several laboratories as to the mechanism of action of pyridoxal phosphate in phosphorylase. In view of the complex role of phosphorylase in the regulation of glycogen metabolism, the chapter focuses on the phosphorylase system. The interconversion reactions of the two forms of phosphorylase catalyzed by phosphorylase b kinase and phosphorylase phosphatase are briefly reviewed. The chapter evaluates the role of vitamin B6 in carbohydrate metabolism in general.

Ca2+ and Sr2+ activation: Comparison of cardiac and skeletal muscle contraction models

The mechanism of contraction in rabbit fast-twitch, and bovine and rabbit cardiac muscle was examined using functionally skinned fibers, ATPase activity of myofibrils, and cardiac or skeletal troponintropomyosin regulated actin heavy meromyosin. The Ca2+ and Sr2+ activation properties for the different measures of contraction were evaluated. (1) Tension in rabbit and bovine cardiac skinned fibers and rabbit cardiac myofibrillar ATPase were activated equally well by either Ca2+ or Sr2+. By contrast, rabbit adductor magnus (fast-twitch) skinned fibers required substantially higher [Sr2+] than [Ca2+] for activation, as did rabbit myofibrils from back muscle (fast-twitch). (2) Substantially more Sr2+ than Ca2+ was also required for activation of skeletal muscle actin heavy meromyosin ATPase, controlled by either the skeletal or cardiac troponin-tropomyosin complex, similar to the activation of fast-twitch muscle. (3) The absence of correlation between the divalent cation selectivity properties of actin heavy meromyosin ATPase controlled by cardiac troponin-tropomyosin and cardiac muscle tension or myofibrillar ATPase activation by Ca2+ and Sr2+ suggests that troponin, if primarily responsible for the activation of cardiac muscle, has very different in vivo and in vitro binding properties. (4) The close correlation between percentage of maximal Ca2+- and Sr2+-activated myofibrillar ATPase and tension in skinned fibers strongly justifies the use of myofibrillar ATPase, in contrast to a reconstituted troponin-tropomyosin actin heavy meromyosin ATPase system, as a biochemical measure of contraction.