Giuseppina Ferri

Subunit structure and activity of glyceraldehyde-3-phosphate dehydrogenase from spinach chloroplasts

Glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate : NADP+ oxidoreductase (phosphorylating), EC 1.2.1.13) from spinach chloroplasts is a polymeric protein of approx. 600,000 daltons and sodium dodecyl sulphate gel electrophoresis shows that it consists of two subunits of molecular weight 43,000 and 37,000. Comparison of amino acid analyses and tryptic peptide maps indicates that the two subunits have a different primary structure. The native enzyme contains 0.5 mol of NADP+ and 0.5 mol of NAD+ per protomer of 80,000 daltons, no reduced pyridine nucleotides have been detected. Almost complete inactivation is obtained by reaction of two cysteinyl residues per 80,000 daltons with tetrathionate or iodo[14C2]acetic acid; since the same amount of radioactivity is incorporated in the two subunits it is likely that they are both essential for the catalytic activity. Charcoal stripping of native glyceraldehyde-phosphate dehydrogenase produces an apoprotein which still retains most of the enzymatic activity but, unlike the holoenzyme, is gradually inactivated by storage at 4 degrees C and does not react with iodoacetate under the same conditions in which the holoenzyme is completely inactivated.

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.

Structural and functional characterization of three human immunoglobulin κ light chains with different pathological implications

The structural properties of three immunoglobulins light chains: kappa SCI, responsible for light chain deposition disease (Bellotti, V., Stoppini, M., Merlini, G., Zapponi, M.C., Meloni, M.L., Banfi, G. and Ferri, G. (1991) Biochim. Biophys. Acta 1097, 177-182), k INC responsible for light chain amyloidosis (Ferri, G., Stoppini, M., Iadarola, P., Bellotti, V. and Merlini, G. (1989) Biochim. Biophys. Acta 995, 103-108) and the non-pathogenic kappa MOS were analyzed by fluorescence spectroscopy and circular dichroism. Comparative evaluation of the data shows that SCI and MOS have similar stability under different conditions, while the amyloid k INC behaves as a very unstable protein. As calculated from the GdnHCl curves, the midpoint of unfolding transition was 1.35 M for SCI, 1.20 M for MOS and 0.1 M for INC. Analysis of CD spectra evidences that the three proteins conserve their conformation in the range of pH 4-8. Change in temperature at pH 4.0 produces the premature transition of INC (Tm 40 degrees C) with respect to SCI and MOS (Tm 50 degrees C). At this pH both the pathological SCI and INC light chains aggregate at a temperature of 20 degrees C lower than the normal counterpart. The specific kidney deposition of kappa SCI has been evidenced after injection of the 125I labelled light chain into mice. No deposition was detectable in the case of INC and MOS.

Structural characterization of two genetic variants of human serum albumin

In the present paper we report the structural characterization of two genetic mutants of human serum albumin: albumin Vanves, a very rare, electrophoretically fast variant of French origin, and albumin Verona, a slow-migrating variant which is the most frequently observed in Italy and which possesses the same electrophoretic mobility as albumin B. Both variants were isolated from the sera of healthy heterozygous subjects. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to the COOH-terminal region of the molecule (residues 549-585) in both cases. The modified fragments were then isolated on a preparative scale by HPLC and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by HPLC, established the mutation responsible for albumin Vanves as 574 Lys----Asn and the molecular defect of albumin Verona as 570 Glu----Lys, both probably due to point mutations in the structural genes. The amino-acid substitutions found in albumins Verona and Vanves are consistent with the electrophoretic mobilities observed for the native proteins at pH 8.6.

The amino acid substitution in albumin Roma: 321 Glu → Lys

Albumin Roma is an electrophoretically slow moving genetic variant of human serum albumin found in 22 unrelated families. The protein was isolated from the serum of a healthy, heterozygous subject. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to fragment CNBr IV (residues 299–329). This fragment was isolated on a preparative scale by RP‐HPLC and subjected to tryptic digestion. Sequential analysis of two abnormal tryptic peptides, purified by RP‐HPLC, revealed that the variant arises from the substitution of glutamic acid 321 by lysine. This amino acid replacement, probably resulting from a point mutation in the structural gene, causes a change in the net charge of +2 units which is in keeping with the decreased electrophoretic mobility of the native protein.

The molecular defect of albumin Tagliacozzo: 313 Lys+Asn

Albumin Tagliacozzo is a fast‐moving genetic variant of human serum albumin found in 19 unrelated families. The protein was isolated from the serum of a heterozygous healthy subject. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to CNBr fragment IV (residues 299–329). This fragment was isolated on a preparative scale and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by RP‐HPLC, revealed the variant was caused by 313 Lys → Asn substitution, probably due to a point mutation in the structural gene. The lack of a lysine residue accounts for the electrophoretic behavior of albumin Tagliacozzo.

Two alloalbumins with identical electrophoretic mobility are produced by differently charged amino acid substitutions

We describe the amino acid substitutions of albumins Sondrio and Paris 2, two slow moving variants of human serum albumin, which show an identical electrophoretic mobility on cellulose acetate at three different pH values. These variants have been found in several instances in a wide geographic area including Northern Italy and France. Both alloalbumins were isolated from the sera of heterozygous subjects. Isoelectric focusing analysis of CNBr fragments from the purified variants allowed us to localize the mutation of albumin Sondrio in fragment CNBr V (residues 330-446) and that of albumin Paris 2 in CNBr VII (residues 549-585). Sequential analysis of the variant CNBr VII established the molecular defect of albumin Paris 2 as 563 Asp----Asn. Fragments CNBr V from normal and Sondrio albumins were isolated on a preparative scale and subjected to tryptic and V8 proteinase digestion. Sequence determination of the abnormal tryptic and V8 peptides revealed that the variant arises from the substitution of glutamic acid 333 by lysine. Thus, a +1 change in the C-terminal region of the albumin molecule produces a variant with the same electrophoretic mobility as an alloalbumin with a +2 substitution in the central domain, suggesting a higher degree of exposure to the solvent of the C-terminal tailpiece. Both amino acid substitutions are consistent with a G----A transition in the first position of the corresponding codon in the structural gene.

Proteolytic and partial sequencing studies of the bifunctional dihydrofolate reductase-thymidylate synthase from Daucus carota

The bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota has been further characterized as regards molecular weight, amino acid composition, protease digestion and microsequencing of proteolytic peptides. Data reported in this paper demonstrate that the carrot protein has a calculated Mr of 124000 thus indicating that, contrarily to what has previously been suggested, it occurs as a dimer of identical subunits. Results of partial amino acid microsequencing show the presence of sequences highly homologous with those of the active sites of both DHFR and TS from other organisms confirming, at the structural level, the bifunctional nature of the carrot protein. As in the case of Leishmania tropica DHFR-TS, incubation of the carrot protein with V8 protease led to a rapid loss of TS activity while retaining that of DHFR. However the pattern of proteolysis did not allow to establish whether the sequence of domains is DHFR-TS as in Leishmania, or vice versa. Low homology of other amino acid sequences, as judged by computer analysis, and absence of common epitopes indicate an apparent divergence between carrot and leishmanian proteins.

The molecular defect of albumin Castel di Sangro: 536 Lys å Glu

Albumin Castel di Sangro is a rare fast-moving variant of human serum albumin which has been discovered in heterozygous form in the serum of an 85-year-old woman living in Castel di Sangro (Abruzzo, Italy). Isoelectric focusing analysis of CNBr fragments from the purified variant allowed us to localize the mutation in fragment CNBr VI (residues 447-548). This fragment was isolated on a preparative scale and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by reverse-phase and cation-exchange HPLC, revealed that the variant arises from the substitution of lysine 536 by glutamic acid. This amino acid replacement, probably due to a single-base substitution in the structural gene, causes a change in the net charge of -2 units, which is in keeping with both the increased electrophoretic mobility of the native protein and the isoelectric point of the modified CNBr fragment.

Use of an Anti‐Idiotypic Monoclonal Antibody in Studying Amyloidogenic Light Chains in Cells, Urine and Fibrils: Pathophysiology and Clinical Implications

A monoclonal anti‐idiotype antibody (IgG1k MoAb 3B11D4) raised against the amyloidogenic DEPλ chain dimer binds a conformational idiotope also present on the monoclonal DEP IgA immunoglobulin. MoAb 3B11D4 does not recognize the reduced and alkylated λ chain monomers, nor the 15‐17‐kDa fibrillar light chain fragments which have the same N‐terminal sequence of the urinary light chains. The lack of about 70 ammo acid residues ofthe C terminal of the protein prevents the formation of the self‐limiting dimer and may facilitate the deposition of the fragments into amyloid fibrils. MoAb 3B11D4 recognizes the plasma cell clone in bone marrow and 9% of circulating B lymphocytes. Panning experiments demonstrate that this antibody has the capability to selectively eliminate the idiotvpe positive cells from peripheral blood. Antibodies with these characteristics could become a useful tool for better understanding the pathogenesis of the disease and for new therapeutic options.