Determination of the intracellular protein thiol distribution of hepatocytes using monobromobimane derivatisation of intact cells and isolated subcellular fractions

The derivatisation of intact rat hepatocytes with monobromobimane resulted in rapid labelling of accessible protein thiols in several subcellular fractions. The derivatisation procedure did not cause acute cytotoxicity, nor did it alter the buoyant densities of the fractions or their gross protein compositions. Quantitation of the fluorescence irreversibly associated with the fractions demonstrated considerable intracellular heterogeneity in this pool of thiols. Values were highest in cytosol (ca. 90 nmol/mg protein), intermediate in microsomes (ca. 65 nmol/mg protein) and mitochondria (ca. 45 nmol/mg protein) and lowest in a crude fraction containing both nuclei and plasma membrane (ca. 35 nmol/mg protein). Similar values were obtained from microsomes and cytosol derivatised after fractionation but there were significant increases of ca. 100% in corresponding values from isolated mitochondria and the nuclear/plasma membrane fraction. These results are discussed in terms of the dynamic fluxes in monobromobimane protein thiols during fractionation and the applicability of this noninvasive method to studies of the mechanism(s) of toxicity of reactive xenobiotics and the role(s) of protein thiols in normal cellular function.     

Detection and quantification of patulin and griseofulvin by high pressure liquid chromatography in different strains of Penicillium griseofulvum Dierckx

Patulin and griseofulvin production by twelve strains ofPenicillium griseofulvum Dierckx, eleven of which were isolated from pistachio (Pistacia vera) nuts and the other was supplied by the Spanish Collection of Type Culture, was investigated. Six strains of the eleven isolated had ability to produce patulin and griseofulvin in Yes medium. All the strains studied had no ability to produce patulin in Wickerham medium. Griseofulvin production was significant in both media but higher in Wickerham. These metabolites were separated and determined in the chloroform extracts of cultures by high performance liquid chromatography with ultraviolet detection. The best conditions were: acetonitrile — water (45∶55) as mobile phase, a flow rate of 2.0 mL/min and a μ Bondapack C₁₈ column.     

Effect of intraarterial somatostatin infusion on SMA blood flow

Experiments were carried out on anaesthetized cats to study the effect of somatostatin on the mesenteric circulation. Intraarterial infusions of somatostatin were applied into the superior mesenteric artery. The effect of atropine, propranolol and phentolamine were investigated. Catecholamine release or uptake of the mesenteric vascular bed during somatostatin infusions were also measured. We found dose dependent vasodilatory effect of somatostatin on the mesenteric vasculature that was not influenced by atropine and by the adrenoreceptors or by denervation. A direct effect of somatostatin on the vascular smooth muscle membrane is assumed.     

Aldosterone nuclear receptors in kidneys of chick embryo

We have studied the properties of the nuclear receptors for aldosterone in kidneys of chick embryo. Aliquots of 0.4 M KCl nuclear extracts were incubated with [3H]aldosterone with or without 1 microM RU28362, a potent glucocorticoid analog. Scatchard analyses of binding data revealed two classes of binding sites with Ka of 0.26 and 0.03 X 10(9) M-1 and Nmax of 330 fmol and 620 fmol/mg DNA respectively. In presence of RU28362, however, we observed only a single class of binding sites with a Ka of 1.02 X 10(8) M-1 and a Nmax of 90 fmol/mg DNA. Competition studies performed in presence of RU28362 showed that aldosterone was the more effective competitor followed by corticosterone, progesterone, deoxycorticosterone, dexamethasone, cortisol, triamcinolone acetonide and cortisone. The nuclear complexes had a sedimentation coefficient in the area of 8 S which changed to 4-5 S in the presence of 0.4 M KCl. This effect of KCl was prevented by the addition of 10 mM sodium molybdate. Always in the presence of the glucocorticoid analog, by DEAE-c chromatography we observed a major specific aldosterone-binding fraction which was eluted with 0.2 M KCl. This fraction sedimented at 8.4 S in the absence of sodium molybdate and KCl. In the absence of RU28362, DNA-c columns retained only a small portion of the nuclear complexes which were eluted with KCl. These complexes sedimented, on sucrose gradient, at 4.6 and 3.1 S, whereas those which did not bind to DNA-c had a sedimentation coefficient of 8 S. In the presence of RU28362, the majority of bound [3H]aldosterone remained in the column flow-through fraction; when this fraction was further analyzed on DEAE-c, complexes were eluted with 0.2 and 0.3 M KCl. These data indicate that nuclear receptors for aldosterone are present in small number in kidneys of chick embryo and that they are mostly in the 8 S form.     

Sequence of the human erythrocyte phosphoglycerate mutase by microsequencer and mass spectrometry

We have previously reported the isolation in pure form of the human erythrocyte phosphoglycerate mutase isozyme B. We now report the sequence of the whole protein and the identification of its N-terminal blocking group. The protein tryptic peptides of phosphoglycerate mutase isozyme B were isolated by high performance liquid chromatography and their sequence determined by microsequencing. The sequence and the nature of the blocking group of the N-terminal tryptic peptide was shown to be N-acetyl-Ala-Ala-Tyr-Lys by mass spectrometry. Overlaps of the tryptic peptides were obtained by studying the V8 Staphylococcus aureus protease peptides of the aminoethylated phosphoglycerate mutase isozyme B either by microsequencing or by mass spectrometry. The procedure used allowed us to obtain the sequence on a very small amount of material and in a short period of time. Our data agree well with those derived from the cDNA nucleotide sequence described by Sakoda et al. (Sakoda, S., Shanske, S., DiMauro, S., and Schon, E. A. (1988) J. Biol. Chem. 263, 16899-16905). In addition, our data directly indicate that the initiation codon does not introduce a methionine as N-terminal amino acid and allowed the identification of the acetyl N-terminal group.     

Structural characterization of the human B lymphocyte-restricted differentiation antigen CD22. Comparison with CD21 (complement receptor type 2/Epstein-Barr virus receptor)

CD22 and CD21 are glycoproteins primarily expressed on normal and neoplastic human B cells. The surface expression of these two molecules parallel each other during normal B cell differentiation, and the reported relative mobilities for CD22 and CD21 are 130/140 kDa and 140 kDa, respectively. Herein we present a detailed analysis of the biosynthesis and structure of CD22 and also compare it directly to CD21. Electrophoresis under reducing and nonreducing conditions suggested that CD22 and CD21 may have similarities in intra-chain disulfide bond formation. Biosynthesis and processing of CD22 and CD21 were very similar with respect to kinetics and post-translational modification, and both could be phosphorylated. However, endoglycosidase digestion (using N-glycanase and endoglycosidase H) and peptide mapping (using V8 protease and N-chlorosuccinimide) strongly suggested that CD22 and CD21 are distinct gene products.