Jan-Olof Jeppsson

Folic acid-an innocuous means to reduce plasma homocysteine

With an improved highly reproducible method, we measured total plasma homocysteine (free plus protein-bound) and related amino acids in the fasting state in healthy subjects, before and after treatment with co-factors for homocysteine metabolism: 1 mg cyanocobalamin (n = 14), 5 mg folic acid (n = 13) or 40 mg pyridoxine hydrochloride (n = 15) daily for 14 days. Cyanocobalamin and pyridoxine hydrochloride had no effects on plasma levels of amino acids, but folic acid had a considerable homocysteine-lowering effect. Total plasma homocysteine was reduced in all but two subjects, from 19.9 +/- 4.4 (mean +/- SEM) to 9.5 +/- 1.0 mumol/l (-52%, p less than 0.01). We propose that folic acid in excess acts by enhancing the remethylation of homocysteine to methionine. The finding confirms a previous report by us. Since homocysteine is considered to be an atherogenic amino acid and recent reports suggest that mild to moderate homocysteinaemia is also associated with premature vascular disease, treatment with folic acid might be of use as prophylaxis.

Amino acid substitution Glu→Lys in α1-antitrypsin PiZ

ctl -Antitrypsin (CX, -AT) is the major protease inhibitor (pi) in plasma. More than 20 alleles contribute to the genetic polymorphism [l] . Most alleles give normal cq -AT concentration in plasma. The PiZ allele is consistent with reduced czl -AT level in plasma and decreased electrophoretic mobility at pH 8.6. An amino acid substitution, lack of sialic acid or loss of a complete carbohydrate chain have been proposed as explanation for the atypical electrophoretie mobility. We have earlier reported that the Z-protein contains l-2 residues less sialic acid but almost normal content of the other carbohydrates [2]. Fingerprint and amino acid analysis of a corresponding CNBr-fragment of 63 amino acids from M and Z protein give clear evidence for an amino acid substitution, glutamic acid to lysine. The carbohydrates are attached to the same CNBr-fragment and it is still obscure how this amino acid substitution can interfere with glycosylation and completeness of the carbohydrate chain.

Characterization of α1-Antitrypsin in the Inclusion Bodies from the Liver in α1-Antitrypsin Deficiency

Abstract α1-antitrypsin was isolated from periodic acidSchiffpositive inclusion bodies from the hepatocytes of patients with α1-antitrypsin deficiency and further purified to enable more detailed chemical analysis. Amino acid and cyanogen bromide fragmentation studies showed a close similarity between hepatic and serum (PiMM) antitrypsin in contrast to the carbohydrate analysis, which revealed markedly deficient glycosylation of hepatic antitrypsin. A complete lack of sialic acid and a relative deficiency of all other carbohydrate components could fully explain the difference of approximately 6000 daltons in molecular size between the two proteins. The accumulation of hepatic globules is probably related to the physical properties of the defective antitrypsin, which include marked insolubility and tendency toward aggregation. The results strongly suggest an abnormal amino acid sequence in the peptide chain of the deficient antitrypsin. The interference with glycosylation may be related to steric hindrance...

Human α1-antitrypsin: carbohydrate attachment and sequence homology

Human err-antitrypsin (or-AT) has 3 carbohydrate sidechains [ 11. The attachment point of one of these sidechains has been determined in conjunction with the amino acid sequence of the C-terminal third of the molecule [2]. We now provide further sequence data which define the attachment points of the other 2 carbohydrate sidechains. These data also show the extent of the sequence homology with antithrombinIII and ovalbumin, and provide support for a single reactive centre situated near the C-terminus. tryptic digestion in 2 M guanidine hydrochloride in 0.1 M NI&HCOa buffer, pH 8.0 at 37’C for 15 h with an enzyme to substrate ratio of 1: 10.

Thin-layer isoelectric focusing for haemoglobin screening and its application to haemoglobin Malmö.

Abstract A thin-layer technique for isoelectric focusing of proteins in polyacrylamide gel was developed and applied to various haemoglobins. Small differences in charge not detectable by conventional electrophoretic techniques were revealed. Thus Hb Malmo ( α 2 β 2 97 His → Gln ) could be separated from Hb-A, and Hb-F I from Hb-F II . The method may preferably replace conventional electrophoretic techniques in clinical work.

Characterization of the predominant basic protein in human seminal plasma, one cleavage product of the major seminal vesicle protein

From liquefied human seminal plasma, we purified the predominant basic protein which appears following liquefaction of coagulated semen. The protein was purified in the presence of di-isopropylfluorophosphate to retard its degradation. Heparin-Sepharose chromatography was followed by gel filtration (Biogel P 60) and by fast performance liquid chromatography on a reversed phase column (C8). The basic protein is a single polypeptide chain with an apparent molecular mass of 12.8 kDa, and has a pI value between that of trypsinogen (9.3) and cytochrome C (10.3). The protein contains no carbohydrate, is rich in histidine, glutamate, and lysine, but is devoid of both cysteine and methionine. The amino-terminal portion of the protein sequence is unique: H N K Q E G R D H D K S K G H F H R V V I H H K G G K A H R G-. A specific rabbit antiserum was raised against the 12.8 kDa basic protein. The protein was found to be unique to seminal plasma among all extracellular fluids examined. Three immunologically related 52 kDa, 71 kDa, and 76 kDa proteins were identified in seminal vesicle secretion when it had been reduced. Prostatic enzyme(s) degraded these proteins to the 12.8 kDa basic protein and several other basic proteins with apparent molecular masses below 18 kDa.

Isolation and characterization of two minor fractions of α1,-antitrypsin by high-performance liquid chromatographic chromatofocusing

alpha 1-Antitrypsin is a glycoprotein that separates into five electrophoretic fractions, viz. M2, M4, M6, M7 and M8. Con A-Sepharose separates the protein into three fractions according to the branching degree of the three oligosaccharide chains. The Con A affinity is identical for M4 and M7 and for M6 and M8. Within each pair the proteins were isolated by rapid chromatofocusing. The M7 and M8 have the same carbohydrate structure as the major M4 and M6 respectively, but have lost the first five N-terminal amino acids (Glu-Asp-Pro-Glu-Gly) as compared to the majority of the protein.

Amino acid sequence of the predominant basic protein in human seminal plasma

The predominant basic protein in liquefied human seminal plasma is the major degradation product of the gel‐forming protein secreted by the seminal vesicles. The amino acid sequence of this basic protein is presented. The basic protein contains 52 amino acid residues. It is devoid of cysteine, methionine, tryptophan, and leucine, but contains seven histidine residues located in the NH2‐terminal half of the molecule. The calculated M r of 5753 is in close agreement with that obtained from gel filtration in guanidine‐HCl on Sephacryl S‐200(M r = 6000).

Thin layer electrofocusing followed by electrophoresis in antibody containing gel.

After thin layer electrofocusing in polyacrylamide gel electrophoresis was performed in antibody containing polyacrylamide agarose gel. The technique combines the high resolution of polyacrylamide gel electrofocusing with the high sensitivity of immunologic identification that gives semiquantitative information. The technique is demonstrated on multiple molecular forms of salivary amylase.

Ion-exchange chromatography of physiological sulphur amino acids on a highly crosslinked resin

Abstract Improved resolution of sulphur amino acids and related compounds in physiological fluids is obtained on a new Jeol resin, Type RC-2, with 10 % crosslinkage. The relatively high degree of crosslinking with correspondingly long elution times is neutralized by using more periods of high temperature and increasing the number of buffers from four to six for a two-column system. The fully automatic procedure is carried out for 12 h with a mean coefficient of variation of 1 %. A 70 min programme is also developed for the quantitation of cystine and related compounds obtained from treatment of cystinuria with penicillamine.