Some properties of a homocarnosine-carnosine synthetase isolated from rat brain

—An enzyme from rat brain catalysing the synthesis of the histidine-containing dipeptides carnosine and homocarnosine (l .-histidine: β-alanine ligase (AMP) [EC 6.3.2.11]) was purified about 30-40-fold from a 100,000 g supernatant. Assays were conducted by measuring the incorporation of L-[14C]histidine into carnosine and homocarnosine isolated by paper electrophoresis from the incubation mixture. The ratios of specific activities for the formation of carnosine and homocarnosine were not significantly different for the various purification steps. This was taken as evidence of one enzyme synthesizing both dipeptides. In studying the properties of this enzyme, a pH optimum of 7.4 was shown for carnosine synthesis. The concentrations of amino acid substrates giving maximal synthesis of both dipeptides were in the physiological range found for rat brain. An apparent requirement for ATP, Mg²⁺, and DPN was seen for dipeptide synthesis. A substrate dependent, enzymecatalysed ³²PPi-ATP exchange reaction was observed, suggesting the formation of an aminoacyl-AMP intermediate. Certain other nucleoside triphosphates could substitute for the ATP; this effect showed a specificity toward the dipeptide being synthesized. The apparent requirement for DPN was quite specific, with a number of related compounds having no effect. The stoichiometry of enzyme-catalysed carnosine synthesis was studied. A one to one relationship between carnosine formed and ATP hydrolysed was demonstrated. However, the ratio between carnosine synthesized and DPN hydrolysed was about 6 to 1, indicating a catalytic role for the DPN. The breakdown of DPN did not occur with enzyme alone but was dependent on the presence of substrate.     

A new species of Croton (Euphorbiaceae) widespread in Mexico

Croton rzedowskii is described, the type being from San Luis Potosí. The species ranges from San Luis Potosi westward to Zacatecas, thence southeastward to Oaxaca.     

Morphological alterations in mammalian neurons in vitro in response to hypertonic solutions

Summary Neurons in cultures of central nervous tissue exhibited marked structural changes when exposed to hypertonic solutions. Cellular reactions were described in living neurons as well as after fixation and staining in preparations observed with both the light and electron microscope. The structures involved in these changes were mainly the nucleolus, the nucleus and the Nissl substance.Nucleolus In living neurons, observed with phase contrast optics, the nucleolus became invisible in hypertonic medium. This change occurred within a few seconds, and it was reversible when the cells were brought back to isotonic solutions. Fixation of the cells while exposed to hypertonic solution caused the nucleolus to reappear as a granular body. In stained preparations it appeared as a more irregular body in contrast to the smoothly outlined nucleolus in normal cells. In electron microscopic preparations of neurons which were fixed while exposed to hypertonic solutions the nucleolus was visible only as nucleolar shadow, overlaid by a few small irregular bodies of higher electron density than other nuclear contents.Nucleus The nuclear membrane of living neurons exposed to hypertonic media lost much of its sharp definition and became rather hazy in outline. The nuclear diameter increased about 10% in hypertonic medium, and the nuclear space became somewhat denser when observed with the phase contrast microscope. In Nissl stained preparations the nuclear space was filled with many small granular or rod-shaped bodies in contrast to the clear vesicular appearance of the nuclei of untreated cells. In electron microscopic preparations the nuclear space exhibited a spotty appearance due to the presence of electron dense and light areas.Nissl Substance In living neurons immersed in hypertonic solutions the Nissl substance showed a slight increase in phase density, especially after repeated changes between hypertonic and isotonic solutions. Sometimes a distinct striation in the Nissl substance appeared. In Nissl stained preparations there was no marked change observed in comparison with normal cells. However, in the electron microscope, the Nissl substance of hypertonically treated cells exhibited a marked structural change. The membrane-bound spaces of the endoplasmic reticulum assumed a rather precise orientation parallel to the cell membrane so that in extreme cases a concentric arrangement of endoplasmic cisternae was observed. The normal arrangement of ribosomal granules in rosettes and clusters became disturbed and the granules were more uniformly distributed.The cells as whole units showed a distinct shrinkage in hypertonic solution which may account for the more crowded appearance of various organelles such as mitochondria and Golgi complexes. There was also a marked increase in agranular reticulum profiles and small membrane bound vesicles in treated cells. Vacuoles appeared frequently in the cytoplasm of treated cells; they disappeared upon re-immersion in isotonic medium.This investigation was supported by USPHS Grants NB 03114-04, NB 00690-11 and 5 T 1 GM 495 from the National Institutes of Health, Bethesda, Maryland.Acknowledgement. Mrs. Eleanor W. Morris and Mr. Edwin E. Pitsinger, Jr. gave indispensible aid with the management of the cultures and with photographic procedures.     

AN ANALYSIS OF MYOGENESIS BY THE USE OF FLUORESCENT ANTIMYOSIN

Antibodies against myosin of adult chicken skeletal muscle were labelled with fluorescein and used as staining reagents to analyze the development of trunk myoblasts in the chick embryo. Myoblasts from the brachial myotomes were studied in three ways: (a) Specimens were fixed, sectioned, and stained with iron-hematoxylin. (b) Living myoblasts, and myoblasts prepared by glycerol extraction, were teased and examined by phase contrast microscopy. (c) Embryo trunks were treated with fluorescent antimyosin or with a control solution of fluorescent normal globulin, and were examined by fluorescence and phase contrast microscopy. Both glycerol-extracted and fixed materials were used. Cross-striated myofibrils appeared first in stage 16 to 17 embryos in the series studied by antimyosin staining and fluorescence microscopy. Striated myofibrils appeared first in stage 18 to 19 embryos, in the series stained by iron-hematoxylin, and at stage 22 to 23, in the series studied by glycerol extraction and phase contrast microscopy. In each series, myofibrils without apparent cross-striations were detected shortly before cross-striations were observed. Specific staining by antimyosin occurred only in differentiating myoblasts. Within the myoblasts antimyosin staining was confined to the A bands of the slender myofibrils. The following observations suggest that the first delicate striated structure to appear in the early 3 day myoblast was remarkably mature: (1) The sarcomere pattern both in length and in internal detail, was similar to that of adult muscle. (2) The distribution of myosin, as revealed by antimyosin staining, was the same in the embryonic as in the mature myofibril. (3) Glycerol-extracted myoblasts contracted vigorously on exposure to ATP. The changes in sarcomere band pattern were indistinguishable from those occurring during contraction of adult muscle induced by ATP. (4) ATP contraction was blocked by prior antimyosin staining in embryonic myoblasts as in mature muscle. It is suggested that the early myofibril grows laterally as a thin sheet associated with the sarcolemma, and that growth in length occurs in the growth tips of the elongating myoblast.