Giuseppe Lugaro

The oxidation of steroid hormones by fungal laccase in emulsion of water and organic solvents

Abstract It is shown that fungal laccase, a copper-containing oxidase, catalyzes the oxidation by molecular oxygen of steroid hormones with a phenolic group in the A ring. The oxidation was performed in emulsions of water and an organic solvent, where the enzyme was in the aqueous phase, and the steroidal substrates and metabolites were mainly in the organic phase. With several organic solvents the laccase displayed full activity over a long period of time (up to several days). The course of the reaction in this two-phase system, as a function of time, pH, and enzyme concentration, was analyzed by chromatography of the reaction products. Two reaction products were isolated and partially characterized. The advantages of carrying out enzyme reactions in two-phase systems are discussed: one of them is the possibility of performing the transformation of large amounts of steroids in small volumes.

Inhibition of DNA polymerization and DNA transcription to RNA by seminal plasma peptides

An oligopeptide fraction purified from the extracellular compartment of bull semen and strongly interacting with DNA was shown to hinder mononucleotide polymerizations to DNA and RNA in vitro. The fraction, collectively called seminal plasma inhibitor, was active in the endogenous DNA and RNA polymerase reactions of the nuclei from rat hepatocytes and in the analogous nucleotide polymerizations catalyzed by purified enzymes of bacterial origin. The type of the induced inhibition was studied using the RNA polymerase from Escherichia coli as a representative nucleotidyl transferase. In the enzymatic polycondensation of mononucleotides, the seminal plasma inhibitor appeared to exert its effect mainly by a competitive inhibition for the utilization of DNA templates without specificity with respect to the source and the base sequence of DNA. Concavities of the plots of V0/Vi versus the amounts of inhibitor in the nucleotide polymerizing reactions and of the Dixon plots in the assays of RNA polymerase from E. coli suggested that the isolated oligopeptide fraction contained more than one active molecular species with differential effects at low and high doses. Preliminary results on the microheterogeneity of the seminal plasma inhibitor supported this contention.

Evidence for a peptidic factor in spermatozoa inhibiting the ovarian maturation

Abstract The presence in bull spermatozoa of a specific, non-steroidal-inhibiting factor of ovarian muturation is shown. Its extraction and partial purification are reported. The inhibitor is active at the 1 μg level. No linear dose-response correlation was observed. The peptidic nature of the inhibitor is proved by experiments carried out using proteolytic enzymes.

Purification of the human urinary glycoprotein with gastric antisecretory activity by ‘subunit exchange chromatography’

A glycoprotein with marked gastric antisecretory activity (human urinary gastric inhibitor) has been isolated in our laboratory from human urine [ 1,2]. This inhibitor is made of identical subunits whose molecular weight was evaluated to be -15 000 by gel-filtration and disc-gel electrophoresis in the presence of sodium dodecyl sulphate (SDS) and by C-terminal analysis [3]. Preliminary experiments have suggested that the molecular weight of the glycoprotein depends on pH due to a reversible associationdissociation process. It has been shown recently [4-61 that, in the case of an associating-dissociating protein system, the protein subunits immobilized on a solid matrix will interact with the subunits in solution. This interaction can be measured quantitatively and can be used to analyze the association-dissociation properties of the system under various conditions. It also provides a powerful purification tool because of its specificity. The process which has been called ‘subunit exchange chromatography’ has been already applied to various proteins [4-61; the results obtained with the gastric antisecretory glycoprotein are reported here.

Purification of a human urinary glycoprotein with gastric antisecretory activity

Abstract We have isolated from the urine of pregnant women a high molecular weight glycoprotein with marked gastric antisecretory activity. Its carbohydrate content totals 37%, of which 9% is in the form of sialic acid. This carbohydrate moiety is joined, at least partially (more than 40%), glycosidically to the hydroxyl groups of threonine. Mild alkali treatment releases hexosamines, salic acid, hexoses and threonine in a molar ratio of approx. 1:1:2:1 . Ultracentrifugation, gel filtration and disc electrophoresis studies have shown that the integrity of the carbohydrate-protein linkage is essential in preserving the high molecular weight structure of the protein ( s° 20,w = 7.89 ) and its biological activity. On splitting this linkage, the macromolecule dissociates into small inactive subunits having s° 20,w = 1.50 .

Inhibition of gastric acid secretion by a glycoprotein isolated from human urine (human urinary gastric inhibitor)

The gastric antisecretory activity of an inhibitor newly isolated from human urine (Human Urinary Gastric Inhibitor or HUGI) has been studied. HUGI was given intravenously and its activity determined in the following test systems: gastric secretion in the rat with pyloric ligation; gastric secretion in the dog with a Heidenhain pouch stimulated with pentagastrin, histamine and a protein meal; acid secretion by the isolated gastric mucosa of the rat; gastrointestinal motility; bile flow and gall-bladder tone and arterial and venous blood pressure and heart rate. HUGI was found to have marked activity only in the pyloric-ligated rats and in the dogs with Heidenhain pouches stimulated by a protein meal. Particularly in the dog, HUGI (0.1 to 6.4 micrograms/kg, i.v) markedly inhibited gastric secretion, dose-dependently and without changing the plasma gastrin concentration. Negative results were obtained in the other tests, but these results serve to demonstrate that HUGI is an inhibitor well-differentiated from other glycoproteins or peptides with gastric antisecretory activity, such as urogastrone and GIP. The results obtained to date are not sufficient to allow the mechanism of action of HUGI to be defined.

A non-steroidal gametic factor linked to DNA modulates δ-aminolevulinic acid synthase inducibility acting on liver transcriptional and translational processes

The effect of an acidic factor of low molecular weight (about 1,000 daltons), extracted from bovine spermatozoan DNA, on the inducibility of delta-aminolevulinic acid synthase by ethanol during aging in rat has been examined. The increased enzyme inducibility in 600-day old rats is supported by stimulation of transcriptional and translational processes; on the contrary, in 30-day old rats, the higher enzyme values induced by ethanol are significantly decreased after factor treatment. The active factor is strongly DNA-bound in the native spermatozoan DNA. This would imply a possible role in regulating gene expression in vivo.

Purification and properties of a porcine duodenal glycoprotein with gastric antisecretory activity

Abstract 1. 1. A high mol. wt glycoprotein with marked gastric antisecretory activity was isolated from porcine duodenum. 2. 2. The purified glycoprotein appeared to be formed by a single species of subunits whose mol. wt in presence of SDS was 15,000 ± 1500 (Sephadex G-100 gel filtration) or 12,000 ± 1500 (disc gel electrophoresis). 3. 3. The partial C terminal sequence was Phe-Tyr-Leu.OH. 4. 4. The glycoprotein contains 17.5% carbohydrate: 9.4% hexoses, 1.7% fucose, 2.1% sialic acid, 3.3% hexosamines and 0.9% uronic acids. 5. 5. The carbohydrate-protein linkage was alkali stable. 6. 6. In the pylorus ligated rat, the glycoprotein produced a 50% inhibition at a dosage of 220 μg/kg when testing the volume of the gastric secretion and at 90 μg/kg when the acidity was measured.

Purification of cortico-cerebral factor(s) inhibiting the secretion of corticosterone

Abstract The existence of specific cortico-cerebral factor(s) inhibiting the secretion of corticosterone is shown and its partial purification by deproteinization, gel filtration, desalting and ion-exchange chromatography is described. A fraction is obtained which is active in vivo at the dose of 1.5 μg; the estimated purification is better than 10 4 times. Preliminary data relative to the possible chemical nature are also shown.

[Clinical and immunological study in plasma-cellular proliferative disorders].

Immunological investigations in 4 multiple myelomas with bone lesions, in 1 extraosseus myeloma, and in 1 reticulum-cell sarcoma with areas of plasma cellular transformation are described. Serum proteins were studied with immunodiffusion, immunoelectrophoresis and sedimentation constant. All identified serum paraproteins in the myeloma patients appeared to be of the IgG type, but with different electrophoretic mobility. Three cases were classified as «slow», one as «fast» and one as «intermediate» type. In the patient with reticulum-cell sarcoma alterations of the antigenic properties of the IgG immunoglobulins were present and they were compatible with the presence of a paraprotein. The immunoelectrophoretic pattern as well as the quantitative determination with immunoplates of the immunoglobulins yielded results almost directly proportional to the percentage of plasmacells detected through bone marrow aspiration and to the extent of the disease.