Occurrence of nitrite reductase in citrus leaves

Evidence is presented for the presence of nitrite reductasein citrus leaves. The enzyme has a Km for nitrite of 45 mu andis inhibited by cyanide. However, unlike citrus nitrate reductase(l), it is probably not a metalloflavo protein, although itmay be related to iron. In addition to the enzymatic nitrite reduction, non-enzymaticnitrite reduction was present in citrus leaf preparations. Underin vivo assay conditions nitrite reduction in one-month-oldleaves was not inhibited by cyanide, in contrast with three-month-oldleaves in which nitrite reduction was almost completely inhibited.Thus it appears that in very young citrus leaves most of thenitrite reduction is non-enzymatic. ¹ Contribution from The Volcani Center, Agricultural ResearchOrganization, P. O. B. 6, Bet Dagan, Israel. Series 1972.........2256AE. (Received November 28, 1972; )     

Acidic metabolites of phenylalanine in plasma of phenylketonurics

Seven aromatic metabolites of phenylalanine were determined in plasma of 20 patients with classical phenylketonuria by means of capillary gas chromatography. The results obtained showed good correlation with plasma phenylalanine levels. Plasma aromatic acid levels may prove useful in the diagnosis and management of phenylketonuria, as well as in research of this disorder.     

Radiation inactivation of binding sites for high-density lipoproteins in human liver membranes

High-density lipoproteins (HDL) are involved in 'reverse cholesterol transport'. Whether or not cell-surface receptors for HDL exist and participate in this process remains controversial, and part of this controversy has centered on the nature of the HDL binding sites. We therefore used radiation inactivation to determine the molecular mass of the HDL binding sites in human liver membranes in situ. These binding sites, which shared all the characteristics of previously described putative HDL receptors, had a molecular mass of less than 10 kDa, indicating that they are probably not proteins. In addition, the binding of HDL to protein-free liposomes was characterized and was found to display the same affinity (KD = 5 micrograms protein/ml approximately 5.10(-8) M) as that to cell membranes, indicating that HDL binding to cell membranes may not require membrane proteins. These observations highlight an important application of radiation inactivation: the ability to demonstrate that something - in this case, a high-molecular-weight protein that accounts for the majority of the HDL binding activity in human liver membranes - is absent.     

Complete genomic structure and mutational spectrum of PHKA2 in patients with x-linked liver glycogenosis type I and II.

X-linked liver glycogenosis (XLG) is probably the most frequent glycogen-storage disease. XLG can be divided into two subtypes: XLG I, with a deficiency in phosphorylase kinase (PHK) activity in peripheral blood cells and liver; and XLG II, with normal in vitro PHK activity in peripheral blood cells and with variable activity in liver. Both types of XLG are caused by mutations in the same gene, PHKA2, that encodes the regulatory alpha subunit of PHK. To facilitate mutation analysis in PHKA2, we determined its genomic structure. The gene consists of 33 exons, spanning >/=65 kb. By SSCP analysis of the different PHKA2 exons, we identified five new XLG I mutations, one new XLG II mutation, and one mutation present in both a patient with XLG I and a patient with XLG II, bringing the total to 19 XLG I and 12 XLG II mutations. Most XLG I mutations probably lead to truncation or disruption of the PHKA2 protein. In contrast, all XLG II mutations are missense mutations or small in-frame deletions and insertions. These results suggest that the biochemical differences between XLG I and XLG II might be due to the different nature of the disease-causing mutations in PHKA2. XLG I mutations may lead to absence of the alpha subunit, which causes an unstable PHK holoenzyme and deficient enzyme activity, whereas XLG II mutations may lead to in vivo deregulation of PHK, which might be difficult to demonstrate in vitro.     

Stoichiometry of formation of Ruhemann's purple in the ninhydrin reaction

The course and mechanism of reaction of ninhydrin with amines has both bioanalytical and bioorganic significance since the reaction is widely used for analysis of amino groups and serves as a model for several biochemical reactions that occur in metabolism of phosphonic acid derivatives, deamination, transamination, and transpeptidation. In many cases, e.g., with lysine, cysteine, proteins, the yield of the ninhydrin product, Ruhemann's purple, does not correspond exactly to the expected 1 equiv. per amino group. Possible reasons for this apparent nonideal stoichiometry include slow formation, side reactions, hydrolytic, oxidative, and photolytic instability, and interfering color. The origin and contributions of each of these factors are examined.