Role of neuraminic acid in the heterogeneity of alkaline phosphatase in sheep brain

1. Comparative studies of the polyuridylic acid-directed phenylalanine-incorporating activity of cell-free systems derived from rat and chicken livers demonstrated markedly lower activity in the chicken liver system. 2. The chicken liver cell sap contained the factor(s) responsible for this lower activity. Ribosomes from chicken and rat performed equally well in the presence of rat liver cell sap. Chicken liver cell sap, when mixed with rat liver cell sap, caused an inhibition of incorporation of phenylalanine into acid-insoluble material. 3. Though ribosomal preparations and cell sap from both rat and chicken liver degraded polyuridylic acid to some extent, the chicken liver cell sap contained the largest amount of activity. 4. Rat liver cell sap inhibited the nuclease activities of ribosomal preparations, but no such nuclease inhibition could be demonstrated with chicken liver cell sap.     

Histochemical localization of protein-polysaccharides in renal tissue

The purpose of this study was to investigate the distribution of protein-polysaccharides in the glomerular and non-glomerular regions of the nephron. The techniques used include the digestion of kidney slices with specific polysaccharidases: neuraminidase, hyaluronidase, chondroitinase ABC, and collagenase followed by several cytochemical techniques to identify the glycosaminoglycans and glycoproteins at the light and electron microscope levels. Differential staining of hyaluronic acid and sulphated glycosaminoglycans was accomplished with Alcian Blue at pH 2.5 and pH 0.5, respectively. Sialoproteins were stained with Alcian Blue at pH 2.5. The periodic acid Schiff’s reaction technique was employed for the visualization of collagen. At the electron microscope level the polysaccharides were identified with the periodic acid-chromic acid-silver methenamine reaction. Our results indicated that the major polysaccharide components of the glomerular basement membrane were sialoproteins and collagen, with smaller amounts of hyaluronic acid and various sulphated glycosaminoglycans. Hyaluronidase digestion resulted in partial detachment of epithelial processes from the glomerular basement membrane indicating the hyaluronic acid may have a role in the stability of the attachment of these processes. Tubular basement membranes also contain sialoproteins and sulphated glycosaminoglycans but in considerably lower concentrations than the glomerular basement membrane. Bowman’s capsule appears to contain mostly sulphated glycosaminoglycans and has a lower concentration of sialoproteins and hyaluronic acid.     

The renal Na+/Ca2+ exchange system is located exclusively in the distal tubule.

The movement of Ca2+ across the basolateral plasma membrane was determined in purified preparations of this membrane isolated from rabbit proximal and distal convoluted tubules. The ATP-dependent Ca2+ uptake was present in basolateral membranes from both these tubular segments, but the activity was higher in the distal tubules. A very active Na+/Ca2+ exchange system was also demonstrated in the distal-tubular membranes, but in proximal-tubular membranes this exchange system was not demonstrable. The presence of Na+ outside the vesicles gradually inhibited the ATP-dependent Ca2+ uptake in the distal-tubular-membrane preparations, but remained without effect in those from the proximal tubules. The activity of the Na+/Ca2+ exchange system in the distal-tubular membranes was a function of the imposed Na+ gradient. These results suggest that the major differences in the characteristics of Ca2+ transport in the proximal and in the distal tubules are due to the high activity of a Na+/Ca2+ exchange system in the distal tubule and its virtual absence in the proximal tubule.     

Specificity of tyrosine protein kinases of the structurally related receptors for insulin and insulin-like growth factor I: Tyr-containing synthetic polymers as specific inhibitors or substrates

The receptors for insulin and insulin-like growth factor (IGF) I are structurally similar transmembrane proteins. Ligand binding to the extracellular domain of the receptor stimulates its cytoplasmic tyrosine protein kinase which phosphorylates its own beta subunit as well as exogenous substrates. It is believed, from several lines of evidence, that tyrosine-specific protein kinases are mediating some or all of the actions of insulin (or IGF-I). In order to gain insights into the substrate specificity of the structurally related insulin and IGF-I receptor kinases, we have studied the action of highly purified receptors isolated from human placental membranes. Present studies using selected tyrosine-containing polymers have revealed: (i) Polymers such as (Y,A,E)n and (Y-A-E)n inhibit beta subunit autophosphorylation and exogenous substrate phosphorylation by autophosphorylated receptors. (ii) Insulin receptor kinase is at least 10 times more sensitive to these inhibitors than IGF-I receptor kinase. (iii) (Y-A-E)n is approximately 8 times more potent an inhibitor than (Y,A,E)n toward both receptors. (iv) While (E4,Y1)n and (E6,A3,Y1)n are good substrates for both receptor kinases, the ratio of phosphate incorporation into the former to the latter is characteristically high (approximately 4) for the IGF-I receptor and low (approximately 1) for the insulin receptor. These results imply that the substrate specificity and enzymatic action of these two receptor kinases are distinct.     

Cloning of a cDNA encoding the smallest neurofilament protein from the rat

We have cloned a cDNA coding for the smallest rat neurofilament protein. The cDNA is 861 nucleotides long coding for 287 amino acids from the internal alpha-helical region and the carboxy-terminal tail domain of the neurofilament protein. Comparison of the porcine, mouse and rat neurofilament protein sequences shows that the protein is highly conserved (greater than 93% identity). Blot analysis indicates that the cDNA is derived from a single neurofilament gene that codes for two different poly(A)+ mRNA species.     

Spatial phase and frequency in motion capture of random-dot patterns

A square matrix of spots (A) was presented in rapid alternation with an uncorrelated matrix (B). If the square arrays are superimposed spatially one sees random incoherent motion. However, incoherent motion was seen only if the outer edges were exactly aligned. If the outline of matrix A is shifted horizontally by 1 degree in relation to B, then the edges are seen to oscillate to and fro. Surprisingly, all the dots in the matrix were seen to 'adhere' to the edges and to move horizontally (Ramachandran, 1981). We then aligned the edges again to produce incoherent motion and superimposed a sine-wave grating on the pattern. If the grating was moved horizontally then all the spots 'adhered' to it and moved horizontally as well. This illusion ('motion capture') was optimal (a) at a 90 degrees spatial phase shift of the grating; (b) at low spatial frequencies (less than 0.5 cycles); and (c) when the grating was alternated in step with the dot patterns. Density modulated gratings were just as effective. We conclude that the unambiguous motion signal derived from the grating is applied spontaneously to the dots as well.     

Amino acid sequence of retinal transducin at the site ADP-ribosylated by cholera toxin

Transducin was [32P]ADP-ribosylated by cholera toxin in bovine retinal rod outer segments and then partially purified on omega-amino octyl agarose to remove other ADP-ribosylated proteins. Trypsin digestion of the ADP-ribosylated transducin and further purification using boronate-polyacrylamide beads and high performance liquid chromatography yielded a single radiolabeled tetrapeptide, Ser-Arg-Val-Lys. The ADP-ribose is linked to the guanidinium group of arginine.