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1. The specificity of chymosin on immobilized bovine B-chain insulin is studied. 2. Eight sites of hydrolysis are determined.

ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity was shown in the soluble fraction of rat liver micochondria. Two molecular forms (ATPase 1 and 2) were isolated. ATPase 1 has already been studied. The present paper deals with the purification method of ATPase 2 which was achieved by the following steps: (NH4)2SO4 precipitation. DEAE-cellulose chromatography, hydroxyapatite chromatography, Sephadex G100 filtration and AMP-Sepharose affinity chromatography. The purified protein was characterized by bidimensional polyacrylamide gel electrophoresis. Molecular weight evaluated by SDS-polyacrylamide gel electrophoresis and Sephadex G100 gel filtration was found to be 61 500 +/- 3000.

An immuno-modulator fraction (Alva) extracted from an endemic plant, in the south of Madagascar, the Aloe vahombe, significantly protects mice against bacterial, parasitic and fungal infections. Wishing to verify whether the fraction Alva was active in tumour reduction, we studied its effect on the development of experimental fibrosarcoma and melanoma in mice by intravenous and intracutaneous injections and injections directly into the tumour of the immunostimulant fraction. We have observed cures, only in the case of the McC3-1 tumour but it is encouraging to note that under different experimental conditions the rate of growth of tumours in animals which were treated is slower than in those not treated. The Alva fraction is a substance which is hydrosoluble, thermostabile, having a molecular weight exceeding 30 000 and is a polysaccharide. The predominant sugars are glucose and mannose in 3:1 ratio. Preliminary studies of its action seem to indicate that the Alva fraction acts upon non-specific response and could possibly stimulate the phagocyte activity of the peritoneal macrophagus.

A method for isolation of a soluble ATPase from rat liver mitochondria after freeze thaw cycling is described. Two enzymatically active fractions were separated by DEAE-cellulose chromatography (ATPase 1 and ATPase 2). ATPase 1 has been purified 300 fold. ATPase 1 was homogenous as judged by polyacrylamide gel electrophoresis. The optimum pH of the enzyme was 5.8-6.0 and the optimum temperature was 45 degrees C. The enzyme follows Michaelis-Menten kinetics: Km (9 X 10(-4) M), Vmax (23,6 mumoles Pi released X min -1 X mg protein -1). The enzyme hydrolysed nucleoside triphosphates, but was inactive upon nucleoside di and monophosphates, glucose 6-phosphate, phosphoserine, pyrophosphate and glycerol 2-phosphate. In contrast to membrane bound ATPase, cations have no effect on the enzyme activity. Nucleoside di and mono phosphates and glycerol 2 phosphate inhibited competitively the enzyme. The enzyme was not affected by oligomycin, but was stimulated by lactate, 2-mercaptoethanol and dithiothreitol.

When the mice are given a hypodermic infection of unrefined Vahombe extract, the Aloe called Vahombe is a liliaceous plant growing in the South of Madagascar, they are protected against the infection caused by the Klebsiella, a pneumonia vector to man, giving rise to an experimental septicaemia in the mouse. Neither bactericide nor bacteriostatic activity has been detected yet about Aloe extract. The anti-infectious activity is proportional to the dose of extract injected, the protecting power is the greatest when the mice have been treated with Aloe, two or three days previously to the infection due to Klebsiella pneumoniae. We have determined the LD50 (Lethal dose 50) for the check batches (non-treated mice) and for the batches of protected mice. We were able to show that the previous injection developed the resistance to infection, multiplied from thirtyfold to a hundrefold. We have tackled the purification of the substance--made soluble after lyophilisation of the crude extract--by means of filtration with Sephadex G50. It would be the first time, for all we know, that a substance endowed with organism. At present we are proceeding with the purification of the active principle and contemplating trying the protective power upon virus infections as well as upon cancerous or parasitic ones.

The antigenic relationship between the milk fat globule membrane (MFGM) and the hydrophobic fraction of proteose-peptone (HFPP) was demonstrated, using a mono-specific anti-HFPP antibody.

Glutamic acid uptake in Streptococcus salivarius subsp. thermophilus was energy dependent, the source of energy and adaptation to sugar being important to efficiency of uptake. The disaccharides, lactose and sucrose, stimulated uptake, but cells grown in glucose were more active. Optimum temperature was approximately 40 degrees C and pH approximately 7.0. NaCl was strongly inhibitory to the uptake of glutamic acid although not to that of isoleucine. High specificity existed because only L-aspartic acid was inhibitory.

Phosphopeptide and phosphoprotein phosphorylation was studied in rat brain microsomes and rat brain slices which were incubated in the presence of [gamma-32 P] ATP under various experimental conditions. Radioactive phosphoserine was isolated from phosphopeptides and phosphoproteins. Naplus, K+, Mg2+ and cyclic AMP had a stimulating effect on the labelling of phosphopeptides. Ouabain and Ca2+ lowered the level of 32P incorporation into the phosphopeptides. The phosphoproteins behaved similarly to the phosphopeptides except for the potassium effect. Chase experiments showed a faster decrease in the labelling of phosphopeptides than in phosphoproteins. We suggest that both compounds may be involved in active transport phenomena.

We described previously the existence of a soluble ATPase activity in rat liver mitochondria [1]. The purification and catalytic properties have been described [2]. In a continuation of these experiments, we have studied the immunologic and structural properties of one molecular form of this enzyme : ATPase I. We have prepared the antiserum anti-ATPase I and demonstrated the purity of our enzyme preparation by immunodiffusion and immunoelectrophoresis. An immunohistochemical method also confirmed the localization of ATPase I in the soluble fraction of mitochondria. The molecular weight of ATPase I was measured by G 100 Sephadex gel filtration and was found to be 18,400; electrophoresis on polyacrylamide gels gave a value of 18,600. The pHi of ATPase I was found to be 7,2. Amino acid analysis showed high amounts of aspartic acid, glutamic acid, serine and glycine. The molecular weight calculated from the total amino acid residues was found to be 17,000. Alanine is the NH2 terminal amino acid. The peptide maps obtained after degrading ATPase I with cyanogen bromide or trypsin are in accordance with the methionine, lysine and arginine residues we found in the ATPase I molecule. ATPase I does not appear to be a glycoprotein.

Venom from the posterior half of the venom duct of Conus Geographus was toxic to mice but without effect on a crab, and produced a flaccid spasmodic paralysis in mice. The minimum lethal dose for mice ranged from 5 micrograms to 50 micrograms (net weight) of venom per mouse. Toxin was submitted to Sephadex G 25 chromatography and to ultrafiltration. The venom was inactivated by pronase. The data suggested that the toxin was a peptide of molecular weight of about 1 500-2 000.