The secretion of tropoelastin by chick-embryo artery cells.

Chymotryptic fingerprint analyses of tropoelastin a and tropoelastin b demonstrated a very close relationship between these two polypeptides synthesized in a cell-free system under the direction of chick-embryo polyribosomal mRNA. A similar study on tropoelastin polypeptides extracted in their hydroxylated and under-hydroxylated forms from artery cells incubated with [3H]valine in the absence and presence of alpha alpha'-bipyridine or 3,4-dehydroproline confirmed this close relationship and suggested that tropoelastins a and b are likely to be the products of a single gene. Pulse-chase experiments in which the synthesis and secretion of tropoelastin by artery cells were monitored demonstrated that, after a pulse with [3H]proline, the polypeptides rapidly appeared in the medium and the half-time of tropoelastin secretion was approx. 30 min. Further pulse-chase studies, in which [3H]tropoelastin contents of subcellular fractions were determined, showed that rough and smooth microsomal fractions contained maximal amounts of tropoelastin at different times. The quantity of tropoelastin in the smooth-microsomal fraction was always only a small proportion of that in the rough-microsomal fraction, suggesting rapid translocation of the polypeptides to the plasma membrane. Incubation of the cells with 0.1 mM-colchicine did not markedly alter the rate of secretion or the distribution of tropoelastin between the subcellular fractions, whereas when 1 microM-monensin was included in the incubations the polypeptides were retained in the rough microsomal fraction. The results are consistent with the proposal that tropoelastin may follow a pathway of secretion from rough endoplasmic reticulum to the plasma membrane via secretory vesicles.     

Elastin cross-linking in vitro. Studies on factors influencing the formation of desmosines by lysyl oxidase action on tropoelastin.

The formation of isodesmosine and desmosine in vitro by the action of lysyl oxidase on tropoelastin was studied. The synthesis of desmosines occurred in the absence of additional substances. The formation of desmosines was not affected by removal of molecular O2 from the reaction medium nor was it affected by the lack of proline hydroxylation in tropoelastin. However, there was virtually no desmosine formation at 15 degrees C, a temperature not conducive to coacervation, indicating that coacervation is an important prerequisite for cross-linking.     

Protein disulphide-isomerase activity in chick-embryo tissues. Correlation with the biosynthesis of procollagen.

Protein disulphide-isomerase activity was determined in homogenates of chick-embryo tissues. Activities were highest in tissues active in procollagen synthesis and were maximal at the developmental stage of maximal procollagen synthesis. These variations in protein disulphide-isomerase activity correlate closely with those previously observed for protocollagen hydroxylase activities.     

On the intracellular site of biosynthesis of α-tocopherol in Hordeum vulgare

When barley seedlings were exposed to ¹⁴CO₂ for 1–3 hr, ¹⁴C was preferentially incorporated into plastoquinone, but only in low amounts i     

Studies on the site of biosynthesis of acidic glycoproteins of guinea-pig serum

1. Studies were carried out to determine the cellular and subcellular site of biosynthesis of components of fraction I, an alpha-globulin fraction containing acidic glycoproteins isolated from guinea-pig serum. l-[U-(14)C]Leucine or -valine and d-[1-(14)C]glucosamine were used as precursors. 2. A lag of about 10min. occurred before appreciable label appeared in fraction I of serum after injection of leucine or glucosamine. Label in fraction I after 60min. labelling with glucosamine was present almost entirely in hexosamine and sialic acid. 3. Site of synthesis was investigated by studies in vivo up to 17min. after injection of precursor. Particulate subcellular fractions isolated from liver, spleen and kidney or homogenates of the latter two tissues were extracted with Lubrol. Extracts were allowed to react by double diffusion with antisera to fraction I or to subfractions isolated from it, and gels were subsequently subjected to radioautography. With either amino acid or glucosamine as precursor, only extracts of the microsome fraction of liver formed precipitin lines that were appreciably radioactive. 4. The role of the microsome fraction of liver in the synthesis of these glycoproteins was confirmed by immunological studies after incubation of liver slices with leucine or glucosamine. Incorporation of leucine was also investigated in a cell-free microsome system. 5. Material was also precipitated from certain Lubrol extracts of liver microsomes by direct addition of antiserum and its radioactivity measured. Degradation of material thus precipitated and use of heterologous immune systems showed that labelling of precipitin lines represented biosynthesis. 6. A study of extraction procedures suggested that the substances present in the microsome fraction of liver that react with specific antisera are associated with membranous structures. 7. Most or all precipitin lines formed by Lubrol extracts of liver microsomes interacted with precipitin lines given by guinea-pig serum or fraction I, immunological identity being apparent with some lines. The microsome-bound substances thus represent serum glycoproteins or precursors of them. 8. The distribution of label in various tissues and in the protein of subcellular fractions of liver after administration of [(14)C]glucosamine to the guinea pig was also studied. Some variation in results obtained with liver was found depending on the fractionation medium used.     

Studies on the mechanism of oxidative phosphorylation. Catalytic site cooperativity in ATP synthesis

Oxidative phosphorylation catalyzed by bovine heart submitochondrial particles appears to exhibit negative cooperativity with respect to [ADP] and positive cooperativity in catalysis. Eadie-Hofstee plots (v/[S]versus v) of the kinetics of oxidative phosphorylation at the variable ADP concentration range of 1-1200 microM were curvilinear and could be analyzed for two apparent KmADP values differing by one order of magnitude, and two apparent Vmax values. The KmADP values with either NADH or succinate as the respiratory substrate were in the ranges of 10 and 100 microM, and the Vmax values in nmol of ATP formed X min-1 (mg of protein)-1 were, respectively, 500 and 1840 when NADH was the oxidizable substrate, and 550 and 100 when succinate was the energy source. Site-site cooperativity of the ATP synthase, which is a central feature of current theories for the mechanism of oxidative phosphorylation, has been well-documented for ATP hydrolysis by isolated F1-ATPase, but never before demonstrated for mitochondrial ATP synthesis.     

Blockage of tropoelastin secretion by monensin represses tropoelastin synthesis at a pretranslational level in rat smooth muscle cells.

The blockage of protein secretion in the R22 cultured rat aortic smooth muscle cell strain with monensin repressed tropoelastin gene expression at the mRNA level by ca. 50-fold as measured by biosynthetic pulse-labeling, in vitro translation, and hybridization with a tropoelastin genomic DNA probe. These results suggest that tropoelastin gene expression is autoregulated, and they represent the first reported effect of monensin on gene expression.