A TrkA-selective, fast internalizing nerve growth factor-antibody complex induces trophic but not neuritogenic signals.

Nerve growth factor (NGF) is a neurotrophin that induces neuritogenic and trophic signals by binding to TrkA and/or p75 receptors. We report a comparative study of the binding, internalization, and biological activity of NGF versus that of NGF in association with an anti-NGF monoclonal antibody (mAb NGF30), directed against the C termini of NGF. NGF.mAb complexes do not bind p75 effectively but bind TrkA with high affinity. After binding, NGF. mAb complexes stimulate internalization faster and to a larger degree than NGF. NGF.mAb-induced activation of TrkA, Shc, and MAPK is transient compared with NGF-induced activation; yet NGF and NGF. mAb afford identical trophic responses. In contrast, NGF induces Suc-1-associated neurotrophic activating protein phosphorylation and neuritogenic differentiation, but NGF.mAb does not. Thus, an absolute separation of trophic and neuritogenic function is seen for NGF.mAb, suggesting that biological response modifiers of neurotrophins can afford ligands with selected activities.

Restoration of hepatic cytochrome c oxidase activity and expression with acetyl-L-carnitine treatment in spf mice with an ornithine transcarbamylase deficiency.

The sparse fur (spf) mutant mouse, with an X-linked ornithine transcarbamylase deficiency, is a model of congenital hyperammonemia in children. Our earlier studies indicated a deficiency of hepatic carnitine, CoA-SH, acetyl CoA, and ATP in spf mice. We have now studied the effects of a 7-day treatment with acetyl-L-carnitine (ALCAR) in the spf/Y mice on the activity and expression of the respiratory chain enzyme cytochrome c oxidase (COX; EC 1.9.3.1). We found decreased hepatic activity and expression of COX in the untreated hyperammonemic spf/Y mice, which was restored upon ALCAR treatment. Because COX is a mitochondrial membrane protein, we also carried out studies to explain the mechanism of ALCAR through its effect on membrane stability. Our results indicate a decrease of the mitochondrial membrane cholesterol/phospholipid molar ratio (CHOL/PL ratio) with the activity and expression of COX in untreated spf/Y mice. While ALCAR treatment normalized the ratios, it also restored the hepatic ATP production to normal. To study further if there was any effect of ALCAR on the mitochondrial matrix urea cycle enzymes, we measured the activity and expression of mutant ornithine transcarbamylase (OTC; EC 2.1.3.3) and normal carbamyl phosphate synthase-I (CPS-I; EC 6.3.4.16) in spf/Y mice. There was no general effect on the specific activities of the matrix enzymes upon ALCAR treatment, although their mRNA levels were enhanced. Our studies point towards the feasibility of an ALCAR treatment in conjunction with other treatment modalities, e.g. sodium benzoate and/or arginine, to improve the availability of cellular ATP and to counteract the effects of hereditary hyperammonemic syndromes in children.

Progressive decrease of cerebral cytochrome C oxidase activity in sparse-fur mice: role of acetyl-L-carnitine in restoring the ammonia-induced cerebral energy depletion.

Sparse-fur (spf) mice with a deficiency of hepatic ornithine transcarbamylase (OTC) are congenitally hyperammonemic, showing elevated cerebral ammonia and glutamine and depleted levels of energy metabolites. This mouse disorder is akin to the human OTC deficiency, in which neuronal loss and Alzheimer's type II astrocytosis is reported. Reduced cytochrome C oxidase (COX) activity is characteristic of neurodegeneration in Alzheimer's type disorders. We have studied the causal relationship between cerebral COX activity and energy depletion in spf mice. Our results indicate a progressive decrease in the COX activity in various brain regions in spf mice, up to 40 weeks of age, which severely effected the cerebral levels of various energy metabolites. A quantitative estimation of cerebral COX subunit I mRNA also showed a tendency to decrease in spf mice. Short-term acetyl L-carnitine (ALCAR) treatment restored these abnormalities. Our study points out that: (a) ammonia-induced alterations in the cerebral reducing equivalents could cause a decrease in COX activity and its mRNA expression, and (b) ALCAR administration could normalize the cerebral energy metabolism and induce COX mRNA expression and activity.

Purification to homogeneity of mitochondrial acyl coa:glycine n-acyltransferase from human liver.

Mitochondrial acyl CoA:glycine N-acyl transferase (ACGNAT) was purified to homogeneity from adult human liver. It was found to be a monomer of 30 kD, having a pI of 6.8. ACGNAT retained 47% of enzymatic activity at 100 mM NaCl concentration, whereas 21% of the activity was retained with KCl and 32% with K3PO4 at 100 mM concentration as compared to the control. The stability studies revealed no change in activity at 4 degrees C for up to 72 h, 25 degrees C for 4 h and at 37 degrees C for 1 h. The Km values of human ACGNAT for benzoyl CoA, salicyl CoA, isovaleryl CoA and octanoyl CoA were 57.9, 83.7, 124 and 198 mM, respectively, and the corresponding Vmax values were 17.1, 10.1, 7.64 and 3.3 mumol/min/mg protein. The availability of pure human ACGNAT would help in studying the molecular genetics and structural biology of this protein which is important in the detoxification of various endogenous and xenobiotic acyl CoA's.

An immunodetection method for the quantitation of human acyl CoA:glycine N-acyltransferase in biological samples.

An immunochemical method for quantitation of human acyl CoA:glycine N-acyltransferase (ACGNAT) has been developed. A 33 kD ovine ACGNAT was purified to homogeneity and was used to raise polyclonal antibodies in rabbits. These antibodies were then employed for developing a sensitive immunodiagnostic method as they cross-reacted with human ACGNAT. Using this methodology, a linear correlation was seen between specific activity and ACGNAT concentration in an ammonium sulphate precipitation fraction (40-60%) of hepatic tissues of children and adults of 4 h age to 11 year old (r = 0.99, n = 6).

Unusual denaturation properties of vicilin from Cajanus cajan.

Pigeonpea (Cajanus cajan) vicilin (Mr 190 kD) holoprotein contains 2 subunits and the N-terminal amino acid sequence is Gly-Ala-Arg-Val-Asp-Gln-Glu for purified vicilin subunit 1 (Mr 72 kD) and Thr-Thr-Cys-Met-Glu-Ser-Gly for purified vicilin subunit 2 (Mr 57 kD). Circular dichroism spectra of vicilin indicate the occurrence of a predominant beta- pleated sheet structure. The fluorescence studies of vicilin reveal its unusual stability to 8 M urea and 6 M guanidine HCl.

Export of proteins across membranes: the helix reversion hypothesis.

A model is presented which explains the biological role of the leader peptide in protein export. Along the lines of this model, the conformational changes of a protein with environment serves as a general mechanism for translocation. The leader peptide in the cytoplasm takes a hairpin like conformation which reverts to an extended helix upon integration into the membrane. The essential features of this model are in accord with recent results of protein export.

A sensitive and rapid immunochemical method for quantitation of proteins.

A sensitive and rapid ELISA for quantitation of seed globulins is described. This method employs conjugation of pigeon pea (Cajanus cajan) globulin antibodies and the enzyme peroxidase together with dextran. Using this conjugate, proteins as low as 0.1 ng were detected. Dextran conjugate has a ten-fold greater efficiency of quantitating pigeon pea globulins than the commercial goat anti-rabbit IgG conjugate, and is three-fold more efficient than pigeon pea globulin IgG peroxidase conjugate. The method can be conveniently adapted for quantitation of other proteins also.

Biochemical and immunological characterization of rice albumin.

Rice albumin from Oryza sativa (Var. Basmati 370) accounts for about 5% of the total seed proteins. A major fraction of rice albumin has been found to be a glycoprotein which is a monomer of 60 kd having iso-electric point 6.54. When rice albumin is digested with trypsin, it shows the presence of 24 peptides as against 28 peptides which were estimated from its amino acid composition. This indicates the presence of a few peptides which resemble each other in their charge and Rf values. Antibodies against Con A purified rice albumin were affinity purified and were used to quantitate the rice albumin levels during post-anthesis by RIA and ELISA. The latter experiments reveal that maximum albumin is present between 18 and 20 days post-anthesis.

Novel application of quantitative immunoassays for screening seed globulins of cowpea varieties.

Using antibodies raised in rabbits, radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) are standardized for cowpea (var. Pusa Barsati) seed globulins. The RIA, when used to screen three stages of seed development, reveals that maximum globulins are detected at 28 days after flowering. When three different varieties of cowpea are assayed for their globulin content by RIA and ELISA, it is observed that the Bold Grain cowpea has the highest amount of related globulin as compared to two other varieties, namely Pusa Phalgun and Asparagus Bean.