Maria Carla Zapponi

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (NADP): amino acid sequence of the subunits from isoenzyme I and structural relationship with isoenzyme II

The structural relationship between isoenzymes I and II of chloroplast glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NADP+ oxidoreductase (phosphorylating) EC 1.2.1.13) has been established at the protein level. The complete primary structure of subunits A and B of glyceraldehyde-3-phosphate dehydrogenase I from Spinacia oleracea has been determined by sequence analysis of the corresponding tryptic peptides, aligned by fragments derived from cyanogen bromide and Staphylococcus proteinase V8 digestions and by partially sequencing each intact subunit. Subunit A has an Mr of 36,225 and consists of 337 amino acid residues, whilst subunit B (Mr 39,355) consists of 368 residues. The amino acid sequence of subunit B, as determined through direct analysis of the protein, is identical to that recently deduced at cDNA level (Brinkmann et al. (1989) Plant Mol. Biol. 13, 81-94). The two subunits share a common portion of amino acid sequence which differs by 66 amino acid residues. Subunit B has an extra C-terminal sequence of 31 amino acid residues. Chloroplast glyceraldehyde-3-phosphate dehydrogenase II was partially characterized by sequencing the N-terminal portion of the intact protein and some of its tryptic peptides. The sequences of all the examined fragments fit precisely that of the corresponding regions of subunit A from glyceraldehyde-3-phosphate dehydrogenase I.

Amino acid sequence of k Sci, the Bence Jones protein isolated from a patient with light chain deposition disease

Light chain Sci was isolated from the urine of a patient affected by light chain deposition disease with an apparent exclusive localization to the kidney. Sci protein is an intact light chain: it consists of 214 amino acid residues and has an Mr of 23.65. Its complete primary structure has been determined by sequence analysis of the corresponding tryptic peptides and by partially sequencing the intact protein. Sequence comparison shows that Sci protein is strictly related to the light chains of kIIIa family (88% structural identity) which are usually expressed in autoimmune rheumatoid syndromes. Computer graphics model suggests a perturbation in k Sci three-dimensional structure due to the unusual replacement of residues 53 and 77.

Spinach chloroplast glyceraldehyde-3-phosphate dehydrogenase (NADP): Formation of complexes with coenzymes and substrates

Abstract Binding of either NADP or NAD to chloroplast apoglyceraldehyde-3-phosphate dehydrogenase ( d -glyceraldehyde-3-phosphate: NADP + oxidoreductase (phosphorylating), EC 1.2.1.13) is accompanied by the appearance of an absorption band similar to the ‘Racker band’ characteristic of glycolytic glyceraldehyde-3-phosphate dehydrogenase. Concomitant enzyme inactivation and disappearance of the Racker band upon reaction with iodoacetic acid indicate that the same cysteine residues which are essential for catalysis participate in the formation of the protein-coenzyme complex. The stoichiometry of the reaction between the enzyme and either coenzymes (NADP and NAD) and substrates (1,3-diphosphoglyceric acid and glyceraldehyde 3-phosphate) is found to be 8:1 (assuming an enzyme molecular weight of 600 000). Titration of apoglyceraldehyde-3-phosphate dehydrogenase with either NADP or NAD indicates that the binding of the two coenzymes occurs at the same site on the enzyme.

Separation of fragments from human serum albumin and its charged variants by reversed-phase and cation-exchange high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography (RP-HPLC) and ion-exchange chromatography on poly(2-sulphoethylaspartamide)-silica (SCX) were compared as alternative approaches in characterizing charged genetic variants of human serum albumin. The chromatographic behaviour of cyanogen bromide (CNBr), tryptic and V8 protease digests from normal and mutant albumins were examined. The results showed that substituted site-containing CNBr fragments are successfully resolved by RP-HPLC; in most instances SCX and RP-HPLC are equally adequate in identifying the modified tryptic peptides from CNBr fragments; although generally useful, SCX chromatography is specifically needed in all instances where amino acid replacement is occurring in a small hydrophilic tryptic fragment and choosing Staphylococcus aureus V8 protease instead of tryptic digestion is advantageous.

Reaction of rabbit muscle apo-glyceraldehyde-3-p- dehydrogenase with pyridoxal-5′-phosphate

A distinctive feature of glyceraldehyde-3-P-dehydrogenase (GPD) (EC 1.2.1 .12) from skeletal muscle is its ability to crystallize with four molecules of firmly bound NAD” [ 1,2] , the removal of which results in the formation of an apoenzyme with markedly different properties, such as a different conformational state and reaction specificity [3-71 . Chemical modifications carried out comparatively on the holo and the apoenzyme should provide information on the nature of the conformational change and, possibly, help elucidate some features of the coenzyme binding site. We have previously shown [8] that native GPD from rabbit muscle may be preferentially labelled at Lys 191 and 2 12 by pyridoxal-5’-phosphate (PLP) with a concomitant loss of catalytic activity. Here we describe the reaction of PLP with the NAD+ free enzyme from the same source and the identification within the primary structure [9] of the residues labelled.

Reversed-phase high-performance liquid chromatographic separation and partial structural characterization of chloroplast glyceraldehyde-3-phosphate dehydrogenase subunits

Abstract Chloroplast glyceraldehyde-3-phosphate dehydrogenase (mol. wt. 600 kD) is composed of 80-kD protomers each made up of two subunits (37 and 43 kD). These subunits have been isolated, from S-alkylated protein, in a homogeneous form and in suitable quantities for carrying out structural studies by reversed-phase high-performance liquid chromatography (RP-HPLC) on a large pore size (300 A) C18-bonded phase. Treatment with dissociating agents necessary to solubilize the S-carboxymethyl enzyme prior to injection, affects the chromatographic behaviour of the enzyme. Urea or guanidine hydrochloride are inadequate for complete dissociation of the protein, which is only partially eluted as distinct subunits, whilst treatment with sodium dodecyl sulphate allows all the injected material to be obtained as separate subunits. Other RP-HPLC supports have been tested: a small pore (100 A) C18-bonded phase is also suitable for resolution of the subunits. On the contrary, no separation can be achieved using a C3 alkyl support. When chromatographed with gel permeation HPLC using denaturing media, the subunits were coeluted because of the small differences in their molecular weights.

High-performance liquid chromatography of complex mixtures of cyanogen bromide-produced peptides from different proteins

A variety of approaches have been required in order to achieve the resolution of large fragments from cyanogen bromide (CNBr) digests of Inc k chain (an immunoglobulin light chain), human serum albumin (HSA) and four of its mutants. Reversed-phase high-performance liquid chromatography (RP-HPLC) under different conditions failed to resolve the Inc k chain digest; the three CNBr fragments (3.1, 6.7 and 13.7 kDa) were separated in a homogeneous form by gel HPLC. Five of the seven CNBr fragments (ranging from 3.4 to 20.0 kDa) obtained from CNBr cleavage of HSA can be resolved by a single reversed-phase HPLC step; separation of the other two requires modification of the eluent composition. Some structural features of the peptides seem to influence their chromatographic behaviour; by examining the elution patterns from albumin mutants, the sequence-related contribution of single amino acid residues is apparent.

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (NADP+) Reactivity of essential cysteine residues in holo- and apoenzyme

On treatment with iodoacetic acid both native and apo-glyceraldehyde-phosphate dehydrogenase (NADP+) (d-glyceraldehyde-3-phosphate : NADP+ oxidoreductase (phosphorylating), EC 1.2.1.13) from spinach chloroplasts are inactivated following a pseudo-first-order process. The rate of inactivation as well as the affinity for the reagent are lower in apo- than in holoenzyme. NADP is more effective than NAD in raising the reaction rate of apoprotein with iodoacetic acid and also enhances the rate of inactivation of native enzyme. Although kinetic evidence indicates that both in native and apo-glyceraldehyde-phosphate dehydrogenase (NADP+) the loss of activity depends on the reaction of 1 mol iodoacetic acid per active site, a different extent of labelling has been found in the proteins: two and four carboxymethyl residues are incorporated per protomer (80 000 molecular weight) in completely inactivated holo- and apoenzyme, respectively. The reactive cysteine residues are located in different tryptic peptides, as suggested by the finding of two labelled spots on fingerprints of holoenzyme: in addition to these, two other faintly radioactive spots appear in inactivated apoenzyme. Partially inactivated apoprotein has been separated by affinity chromatography into a completely inactive species and a fully active one: the relative amount of these species depends on the extent of inactivation of the sample. The number of alkylated cysteines in the fully inactive apoprotein is found to vary as a function of the inactivation extent, and a minimum of two residues per Mr 80 000 has been found. The two different subunits making up the enzyme (43 000 and 37 000 daltons, respectively) behave asymmetrically during the alkylation process showing that the reaction between glyceraldehyde-phosphate dehydrogenase (NADP+) and iodoacetic acid takes place first at the 37 000-dalton subunit.