Miriam Tauber-Finkelstein

Vitronectin is phosphorylated by a cAMP-dependent protein kinase released by activation of human platelets with thrombin

Activation of freshly isolated human platelets with a physiological stimulant (thrombin) causes them to release a cAMP-dependent protein kinase which specifically phosphorylates one plasma protein (Mr 75000). This protein is immunochemically and biochemically identified as vitronectin (also know as S protein), which was previously implicated in blood clotting, complement function and cell adhesion.

Introduction of an intramolecular crosslink at the active site of glyceraldehyde 3-phosphate dehydrogenase

Abstract Reaction of rabbit muscle apo-glyceraldehyde 3 -phosphate dehydrogenase with one mole of 1, 5-difluoro, 2, 4-dinitrobenzene per mole of enzyme protomer brings about total loss of enzymatic activity and concomitant introduction of covalent intramolecular crosslinks at the active site of the enzyme. Following peptic digestion, a crosslinked couple of peptides was purified and found to have the structure: The crosslinked cysteine and lysine residues, though some 32 amino acid residues apart in the primary sequence, may approach each other to a distance of 5–6 A in the three dimensional structure of the enzyme.

Consecutive use of ω-aminoalkylagaroses. Resolution and purification of clostripain and collagenase from Clostridium histolyticum

Abstract Clostripain and collagenase A were resolved and purified from the culture filtrate of Clostridium histolyticum by consecutive use of two columns from the homologous series of ω-aminoalkylagaroses. Clostripain was extracted by passing the culture filtrate through ω-aminobutylagarose and purified ∼9-fold with 70% overall yield. Collagenase A was then extracted by passing the excluded mixture of proteins through ω-amino-heptylagarose and was purified about 7-fold with an overall yield of 90%. This procedure yields two useful proteolytic enzymes with narrow specificity and also illustrates the potential of homologous series of hydrocarbon-coated agaroses for the development of consecutive fractionators. Such fractionators would separate several proteins out of the same crude extract, thus leading to maximal utilization of expensive or scarcely-available tissues.

cAMP-Triggered Proteolysis of cAMP-Dependent Protein Kinase in Brush Border Membranes

Publisher Summary This chapter discusses KSMP as a regulatory device and biochemical characterization of KSMP. The physiological response of cells to hormones that function via cAMP involves a variety of metabolic reactions; yet cAMP-dependent protein kinase (cAMPdPK) appears to be the major intracellular target for cAMP. The main indication supporting the suggestion that KSMP may be involved in the regulation of either cAMPdPK or a structurally similar kinase lies in the remarkable specificity with which KSMP acts on C. Unlike other proteinases, KSMP degrades C in its native conformation but not if the kinase has been heat-denatured. In a study described in the chapter, among several tissues tested, the KSMP activity was identified only in the brush border membranes of the small intestine and of the kidney, two membranes which are morphologically similar and appear to have several physiological functions in common. The activity of the KSMP could be restored by reconstitution of the apoenzyme with various metal ions. The findings from the study suggested that KSMP is a metalloenzyme and that, judging by the relative metal ion concentration sufficient for restoring the KSMP activity, Zn 2+ is most likely the native cofactor of this proteinase.

Thiolysis of O-2,4-dinitrophenyltyrosines: Spectrophotometric monitoring of the reaction and its use in peptide synthesis

Abstract The thiolytic cleavage of O -2,4-dinitrophenyl (Dnp) derivatives of phenols was applied to the synthesis of tyrosine-containing peptides. This paper describes the preparation and properties of starting materials for such syntheses and illustrates their use in the synthesis of some peptides containing tyrosine at either the C- or N-terminus. A spectrophotometric method for following the thiolytic removal of Dnp groups from O -Dnp-tyrosines was developed and used to establish optimal conditions for quantitative deblockage in aqueous and nonaqueous solvents. The method is based on the fact that upon thiolysis, the colorless solution of O -Dnp-tyrosine ( λ max at 298 nm, pH 8.5) becomes yellow due to the formation of a dinitrophenylated thiol (for S -Dnp-2-mercaptoethanol, λ max at 340 nm, pH 8.5). This gives rise to a difference spectrum with a maximum at 354 nm ( Δϵ M = + 8680 M −1 cm −1 ), a minimum at 298 nm ( Δϵ M = −5900 M −1 cm −1 ) and a crossover point at 318 nm, which is different (in the 290–320 nm range) from the difference spectrum obtained upon thiolysis of N Im -Dnp-histidine. This method provides a useful analytical tool in peptide and polypeptide synthesis as well as in protein chemistry.

Reversible dinitrophenylation of glyceraldehyde 3-phosphate dehydrogenase. Identification of the labeled site

The thiolytic removal of dinitrophenyl (DNP*) groups from sulfhydryls, imidazoles’and phenolic hydroxyls [l] has converted dinitrophenylation into a method for the reversible labeling of these functional groups in proteins. The major advantage of this technique lies in the fact that it combines two useful features: a) the label can be easily removed under conditions which do not cause any irreversible damage to the protein, b) its binding is stable enough to withstand the manipulations required for fragmentation and sequence studies, so that the exact location of the labeled amino acid can be identified. We have recently demonstrated the mildness of the procedure by applying it to GAPD (EC 1.2.1.12) [2] . Stoichiometric dinitrophenylation of a cysteine residue in apo-GAPD inactivated the enzyme and subsequent thiolysis of the label resulted in full restoration of the catalytic activity. We wish to report here the isolation and sequence determination of a pentapeptide fragment from GAPD containing the labeled cysteine.

Monitoring the resolution of proteins and peptides in the course of their electrophoresis: Use in studying the degradative inactivation of cAMP-dependent protein kinase

Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate [ 1,2] has now become one of the most widely used analytical tools in biochemistry for the identification of proteins, for the determination of their molecular weight, and for detecting protein degradations correlated with an altered physiological function (e.g., Zymogen activation, activation and inactivation of polypeptide hormones, removal of ‘anchoring’ or ‘signal’ peptides). After the electrophoretic resolution is completed, the relative migration of the various proteins in the mixture is usually revealed by staining overnight with a solution of Coomassie blue (0.02%) in acetic acid (7%), then removal of the excess staining dye either electrophoretically or by extensive washing. We wish to describe here the usefulness of prelabeling the constituents of a mixture to be analyzed with fluorescamine [3] which makes it possible to monitor continuously (under W light) the resolution of proteins and peptides in the course of their electrophoresis. This is exemplified in the case of the degradative inactivation of catalytic subunit of CAMPdependent protein kinase by a specific membranal proteinase from the brush border membrane of the rat small intestine.

Evidence for an interprotomerial active site in D-glyceraldehyde-3-phosphate dehydrogenase.

, each accomodating one mole- cule of NAD’ as a coenzyme. In its amino acid sequence it is similar to the pig muscle enzyme, whose primary structure has been established [l] . It has been shown [2] that the sulfhydryl group of the cystein residue at position 149* is essential for the catalytic activity of the enzyme. Upon removal of NAD’ this cystein residue can be selectively dinitro- phenylated with concomitant loss of enzymatic activity [3,4]. The unique reactivity of this sulfhy- dry1 was used [S] as an anchoring point for the bifunctional labeling reagent 1, 5-difluoro 2,4-dinitro- benzene (Fz DNB)**, thus introducing crosslinks at the NAD’ binding site of the enzyme. One type of crosslink was formed between the sulfhydryl group of Cys-149 and the e-amino group of Lys-183 [5]. Another type of crosslink connected Cys-149 and Cys-153 [6] . It was shown that the crosslinks were formed within the tetrameric molecule, as there was no change in its sedimentation coefficient as a result of the labeling. The possibility was raised [S] that the crosslinked Cys and Lys residues might,originate

A cAMP-triggered release of a hormone-like peptide

Preparations of the catalytic subunit of cAMP‐dependent protein kinase from rabbit skeletal muscle, which appear to be homogeneous by SDS‐polyacrylamide gel electrophoresis, were often found to contain a hormone‐like factor (HLF) which causes an immediate rise, then a decline of intracellular cAMP in a B‐lymphoma cell line. Active HLF is released when the fractions that contain it in an inactive form are incubated with cAMP prior to chromatography, or passed through an immobilized cAMP column. HLF seems to be a peptide: it loses its cell‐stimulating capability after proteolysis and has an apparent molecular mass of 2.2 – 2.5 kDa.

A periodicity in the response of SJL/J thymocytes to isoproterenol. Simulation by cell lines

Thymocytes from SJL/J mice exhibit a periodicity in their response to hormonal stimulation with isoproterenol. This periodicity (5-9 days) is expressed in large changes in the intensity of the response (peak levels of intracellular cAMP which vary approximately 6-fold), and in the response pattern, i.e., in the occurrence or non-occurrence of an immediate hormone-induced desensitization. In contrast, C57BL/6 thymocytes have a homogeneous response pattern (in all cases there was an immediate desensitization). Their response does change with the same periodicity, but these changes are restricted to the intensity of response (the peak of cAMP levels varies only approximately 2-fold). Using T, B and pre-B shown that these periodic changes may be due to fluctuations in T cell subpopulations in the thymus and possibly also the infiltration of B cells into it. These observations provide a useful system for studying at the cellular and molecular level the known correlation between endocrine disorders and neoplasia in mice. On the other hand, the set of cell lines with such different response patterns to a single hormone may be used for the identification and isolation of additional cellular constituents involved in the cellular response to hormones and its immediate desensitization.