Aspartate carbamoyltransferase from rat liver

1. Aspartate-carbamoyltransferase activity was concentrated from rat-liver preparations. Only l-aspartate, beta-benzyl-l-aspartate and beta-erythro-hydroxy-dl-aspartate were carbamoylated enzymically. The K(m) for l-aspartate and carbamoyl phosphate have been determined by three methods: colorimetric procedure, radioactive assay with [(14)C]aspartate and an assay with [(14)C]carbamoyl phosphate. 2. The K(m) for aspartate has been determined as a function of the pH; the pK of the functional group at the active site of the enzyme, pK(e), was at pH9.0. Enzymic activity was diminished in the presence of N-ethylmaleimide, p-hydroxymercuribenzoate and the heavy metals Ag(+), Hg(2+), or Zn(2+). The inhibitions could be prevented by mercaptoethanol. These findings suggested the association of a thiol group with the enzymic activity. 3. Enzymic activity was also decreased by sodium lauryl sulphate, urea and dioxan. Competitive inhibition (with l-aspartate) was manifested by maleate, succinate, oxaloacetate, beta-erythro-hydroxy-dl-aspartate and beta-benzyl-l-aspartate. The K(i) for most of these inhibitions has been determined. 4. The properties of the liver enzyme are compared with those of Escherichia coli aspartate carbamoyltransferase and the implications of the findings are discussed.     

Inhibition of the orthophosphatase and pyrophosphatase activities of human alkaline-phosphatase preparations

1. Purified human liver and small-intestinal alkaline orthophosphatases release inorganic phosphate at appreciable rates from a variety of organic pyrophosphate substrates. 2. The pyrophosphatase action is inhibited by Mg²⁺ ions at concentrations that activate the hydrolysis of orthophosphate substrates by these enzymes. 3. The results of mixed-substrate experiments, denaturation studies with heat or urea and starch-gel electrophoresis suggest that both orthophosphatase and pyrophosphatase activities are, in each preparation, properties of a single enzyme. 4. Intestinal phosphatase shows greater pyrophosphatase activity relative to orthophosphatase than the liver enzyme.     

Morphology of Liberian Negro deciduous teeth I. Odontometry

An odontometric study of the deciduous teeth of 21 Liberian Negro children is reported. These data were obtained from dental casts alone. Statistical comparisons were made of the mesiodistal diameter alone with two American White populations and the three Japanese populations. Maxillary teeth, with the exception of the cuspids were larger in the Negro sample, while only the Negro mandibular molars were significantly wider. This situation resulted in an alteration of Negro maxillary tooth size order. Comparisons were made with other populations reported in the literature which led us to stress the fact that, as yet, no satisfactory sample of deciduous tooth measurements are available for any population. A discussion of tooth form differences stresses the point that teeth differ in both attributes of form, i.e., in shape as well as in size between the several major racial groups. The processes by which these shape differences are brought about were discussed and it was suggested that differing relative growth rates may well be one cause. An allometric analysis of fetal molar tooth growth showed, again, that a sharp interphase occurs in all teeth, with a subsequent decrease in specific growth rate. This interphase corresponds to the initiation of cuspal calcification.     

Properties of alkaline-phosphatase fractions in extracts of human small intestine

1. The chromatographic and electrophoretic heterogeneity of human-intestinal alkaline-phosphatase activity is described. 2. The phosphatase activity has been divided into two main components, of which certain properties have been compared. The components resemble each other in their Michaelis constants for the hydrolysis of phenyl phosphate and in their behaviour towards certain inhibitors, but differ in their stability to heat. 3. The addition of Mg²⁺ or Ca²⁺ ions to the electrophoresis buffers sharpens the electrophoretic zones. 4. The results presented do not support the existence of more than one alkaline phosphatase in small intestine.     

Monoclonal antibody against a melanosomal protein in melanotic and amelanotic human melanoma cells

BALB/c mice were immunized with tyrosinase, partially purified in two stages from a human melanoma cell line. A hybridoma was obtained which produced monoclonal antibody (MoAb 1C11) reactive with 8/10 melanoma cell lines and 10/10 primary cultures of human melanocytes, neval cells, and melanomas. Immunoreactivity correlated to a certain extent with tyrosinase activity but not with melanin content. No crossreactivity was obtained with neuroblastoma, medulloblastoma, fibroblasts, keratinocytes, lymphoid cells, or murine melanomas. Purification of the antigen directly from cell lysates with a MoAb 1C11 CNBr-Sepharose affinity column gave a green-brown protein of 56 kDa with no detectable tyrosinase activity. This protein was therefore different from 60 kDa active tyrosinase, identified by enzyme activity and Western blotting with a MoAb derived previously (MoAb 5C12). Unlike 5C12, 1C11 reactivity was not destroyed by pretreatment of the antigen with periodate. Immunogold labelling showed that the 1C11-reactive antigen was associated with melanosomes, and there was close correlation between 5C12 and 1C11 reactivity in resistance to trypsin and in staining various melanocytic cell populations. MoAb 1C11 may therefore recognise a polypeptide epitope in a molecule closely linked to melanin biosynthesis.