Search

Bahn, Arthur N. (Northwestern University, Chicago, Ill.), Patrick C. Y. Kung, and James A. Hayashi . Chemical composition and serological analysis of the cell wall of Peptostreptococcus . J. Bacteriol. 91: 1672–1676. 1966.—Chemical and serological analyses were made of the cell wall of Peptostreptococcus to characterize taxonomically this genus of anaerobic streptococci. Cell wall hydrolysates of P. putridus strains 06 and 85, P. intermedius strains 11 and 87, and P. elsdenii strain B-159 were prepared, and the cell wall sugars were measured quantitatively by paper chromatography. Strain 85 contained only glucose, whereas strain 06 contained 93% glucose and 7% mannose. Strain 87 contained only rhamnose, and strain 11 contained approximately equal amounts of glucose and rhamnose. Strain B-159 differed from all the other strains in having a low (3.1%) content of total carbohydrate, consisting of rhamnose, galactose, and glucose. Quantitative amino acid analyses showed that the major amino compounds present in the cell wall were glutamic and aspartic acids, alanine, lysine, muramic acid, glucosamine, and galactosamine. Strains 06 and 85 possessed this complement of amino compounds, but strains 11 and 87 had relatively little aspartic acid. Strain B-159 was markedly different in having a high content of glycine and diaminopimelic acid, with only traces of lysine; it was the only strain in which teichoic acid was found. Serological analyses were made with the use of cell wall extracts as antigenic material and with homologous antisera, as well as streptococcal group antisera for groups A through S. The only strong agglutination was obtained between strain 87 antigen and group C antisera; weak agglutination was obtained with 87 against N, O, and K, and between strain 11 and groups E and F. All other antisera gave negative reactions. It is concluded that strain B-159 does not belong to the genus Peptostreptococcus , that strains 06 and 85 are members of P. putridus , and that strains 11 and 87 may be members of two different genera.

Goldfish retinas incubated with L-glutamate-14C (UL) were found to synthesize γ-aminobutyric acid-14C (GABA-14C) The accumulation of newly synthesized GABA was enhanced by physiological stimulation of the retina with flashing light; and this increase was directly proportional to the logarithm of the light intensity. The total GABA content was also higher in light-stimulated than in dark-adapted retinas, although the glutamate content remained unchanged No differences were found in the cell-free activities of glutamate decarboxylase (EC 4 1.1 15) and GABA-glutamate transaminase (EC 2.6.1.19) extracted from light-stimulated and dark-adapted retinas. These findings, together with other physiological and morphologcal evidence, suggest that GABA plays a functional role in synaptic transmission in the goldfish retina

Normal human immunoglobulin G (IgG) was treated with radioactive N-ethylmaleimide in the absence of a reducing agent but in the presence of a denaturing solvent. The sites of reaction were determined by isolation of the radioactive peptides from thermolytic digests of the heavy and light chains. Six radioactive peptides were purified from the digest of the light chain and ten from that of the heavy chain. When sequenced, all the peptides were identical with peptides that would be predicted for the half-cystine residues involved in disulphide bonds. The specific radioactivity of the peptides indicated that the proportion of the half-cystine residues in the reduced form varied from 0.57 to 2.54%. These results indicate that the disulphide bonds of IgG are not completely oxidized. The estimate of 0.2mol of SH group/mol of IgG (Cecil & Stevenson, 1965; Luks & Connell, 1968) can be accounted for if 0.8% of every half-cystine residue of the intrachain bonds was in the reduced form. Variable cysteine residues as have been described in several myeloma proteins must occur extremely infrequently among the immunoglobulins.

In attempting to evidence the evolution and the structure--function relationships of the ribosome in procaryotes the authors have undertaken a comparative amino acid sequence analysis of ribosomal proteins from Escherichia coli, and Bacillus stearothermophilus and Halobacterium cutirubrum, organisms which differ substantially in their physiological tolerances and taxonomic relationships. Previous structural studies have indicated a high degree of homology in some of the 30 S ribosomal proteins from E. coli and B. stearothermophilus. Reported in this paper is a summary of results on the study of the amino terminal regions of 19 ribosomal proteins E. coli strain Q13 and 21 from B. stearothermophilus strain 10.

A unicellular blue-green alga, Agmenellum quadruplicatum , and a filamentous blue-green alga, Lyngbya lagerheimíi , were grown heterotrophically in dim light with glucose as major source of carbon and possibly energy. The dim-light conditions did not support autotrophic growth. The two blue-green algae appeared to have the same metabolic block, namely an incomplete tricarboxylic acid cycle, as has been found in other obligately phototrophic blue-green algae. Under dim-light conditions, glucose made a greater contribution to cell constituents (amino acids) of A. quadruplicatum and L. lagerheimii than under high-light conditions.

The complete amino acid sequence of wool protein SCMKB-IIIB3 was determined. The peptides used for the sequence work were obtained by peptic and thermolysin digestions and were fractionated by chromatography on DEAE-cellulose, paper chromatography and electrophoresis. The peptides were analysed by dansyl–Edman degradation, mass spectrometry and tritium-labelling of C-terminal residues. The protein consists of 98 residues and has acetylalanine as N-terminal residue and carboxymethylcysteine as C-terminus. It is homologous with protein SCMKB-IIIB2 (Haylett & Swart, 1969). A salient feature of the sequence of protein SCMKB-IIIB3 is three consecutive cysteine residues.

A strain identified as Corynebacterium fascians was found to accumulate extracellular D-alanine from glycerol. Cultural conditions for the accumulation of D-alanine were investigated and, as a result, a yield of 7 g of D-alanine per liter was obtained after a 96-h incubation in a medium containing 5% glycerol, 4% (NH 4 ) 2 HPO 4 , and 0.3% corn steep liquor. Optical purity of D-alanine was dependent upon the concentration of corn steep liquor. At the optimal condition, almost optically pure D-alanine was formed and readily isolated (5 g/liter) from the fermentation broth. The product was not contaminated with any detectable amount of other amino acids, except for glycine which was present at a concentration of less than 1 percent.

Keratan sulphate was isolated from adult intervertebral disc in 90% yield by sequential digestion of the whole tissue with papain, Pronase and Proteus vulgaris chondroitin sulphate lyase. Treatment of this preparation with alkali cleaved a glycosidic bond between N-acetylgalactosamine and threonine and produced, by an alkali-catalysed ‘peeling’ reaction, an unsaturated derivative of N-acetylgalactosamine which reacted as a chromogen in the Morgan–Elson reaction, but remained covalently bonded to the keratan sulphate chain. This derivative was reduced and labelled by alkaline NaB3H4. The substituent at position 3 of N-acetylgalactosamine in the keratan sulphate–protein linkage was identified as a disaccharide, N-acetylneuraminylgalactose, which was isolated from the reaction mixture after alkali treatment.

being identified by two-dimensional electrophoresis [2] and amino acid composition [5]. Tryptic peptides of L20, prepared by incubation at 37°C for 3 hr with a 1% (weight enzyme: weight protein) trypsin solution, were fractionated by gel fil- tration chromatography on a Sephadex G-50 super- fine column (1.5 X 270 cm) with distilled water as the eluant. The resulting peaks, which were monitored at 230 nm, were analysed for amino acid composition with a Durrum D-500 amino acid analyser after hy- drolysis at 110°C for 18 hr with 6 N HCl in vacua. Peptide L20-T4 was eluted soon after the void volume and was well separated from other smaller tryptic pep- tides. The amino-terminal sequence of protein L20 (6 mg) and L20-T4 (2 mg) was determined by automatic Edman degradation (91 using a Beckman Model 890C sequencer with the quadral protein program. The thia- zolinone derivatives, converted to their PTH-deriva- tives, were identified by thin-layer chromatography on silica gel plates as described by Wittmann-Liebold [lo] The thiazolinone or PTH-derivatives were hydrolysed separately with 6 N HCl and Hl [ 1 I] at 130°C for 20 hr, and the amino acid formed was analysed with a Durrum D-500 amino acid analyser. 127

1. The selective isolation of an `active' histidine peptide from reduced and cyanoethylated chymotrypsin-α inhibited with Tos-Phe-CH2Cl (l-1-tosylamido-2-phenylethyl chloromethyl ketone) was obtained with a His(τCm) (Nτ-carboxymethylhistidine) diagonal peptide-mapπng'technique.Performicacapours,usedbetweenthefirstandseconddimensionsofthediagonalpeptmapπng′technique.Performicacapours,usedbetweenthefirstandseconddimensionsofthediagonalpeptmap', resulted in a peracid rearrangement of the alkylated (Tos-Phe-CH2)-histidine-57 residue into an Nτ-carboxymethylhistidine residue. The consequent change in electrophoretic mobility allowed isolation of peptides that contained the `active' histidine. 2. Peptides containing methionine or S-cyanoethylcysteine were oxidized to their sulphones during the treatment. Peptides in which these residues were N-terminal were selectively isolated on the basis of the change in electrophoretic mobility at pH6.5 which was due to the depression of the pK of the terminal amino group by the inductive effect of the sulphonyl group. 3. An attempt to apply the method to subtilisin BPN′ inhibited with l-1-benzyloxycarbonylamido-2-phenylethyl bromomethyl ketone failed to yield a peptide containing Nτ-carboxymethylhistidine, although peptides containing N-terminal methionine were isolated by the procedure.

When cultures of biotin-requiring mutant of Brevibacterium divaricatum NRRL-2311 were treated in early logarithmic phase with penicillin (2–3 units/ml) in a glucose-mineral medium under excess supply of biotin, large amounts of a high-molecular-weight material accumulated in the medium. The phenomenon could not be observed with cultures run in parallel from which penicillin was omitted. Chemical analyses of the excreted material isolated from the media of 1-h and 24-h penicillin-treated cultures, showed that the main constituents were peptidoglycan components of noncross-linked structure bearing both l and d-alanine residues; evidence was also obtained for the occurrence of extractable lipid material, non-amino sugars and organic phosphate. Under identical conditions, the excretion of peptidoglycan could be induced by ampicillin and cloxacillin, respectively, but not by bacitracin. Addition of penicillin to biotin-requiring mutant of M. glutamicus yielded similar results, indicating that the phenomenon was not restricted to the Brev. divaricatum strain. This suggests that excretion of peptidoglycan material by the two biotin-requiring mutants might be the result of two events, (a) a change in the osmotic barrier of the cell and (b) specific inhibition of cross-link formation in peptidoglycan induced by penicillin. Several procedures were examined for the isolation and purification of the peptidoglycan complex excreted by penicillin-treated Brev. mutant. Suitable labelling experiments with l-[U-14C]glutamic acid and analyses of lysozyme digests of the purified fractions, suggest that some of the components might contain murein fragments covalently linked to a polysaccharide portion.

A procedure for detecting the degradation of amino acids by microorganisms is described, and examples of its use in the characterization of bacteria are presented. The procedure involves inoculating a buffered solution of amino acids with a suspension of bacteria, incubating, chromatographing a sample of the suspension, and detecting degradation in terms of absence of ninhydrin-positive spots.

A heptapeptide has been isolated from the active site of Candida utilis transaldolase after chymotryptic digestion. The amino acid sequence of this peptide has been established as: Ile-Lys-Ile-Ala-Ser-Thr-Tyr. The lysine residue is that involved in Schiff base formation with the substrate.

The oligosaccharide units of a type K and a type L IgA immunoglobulin have been examined. The two proteins differed in their content of 6-deoxy-l-galactose and N-acetylneuraminic acid, and in the d-mannose/d-galactose ratio. With glycopeptides prepared from the type K protein, specific glycosidases liberated the N-acetylneuraminic acid and 7-8 residues of 2-acetamido-2-deoxy-d-glucose, and mild acid hydrolysis released most of the 6-deoxy-l-galactose. The type K immunoglobulin appeared to contain 3 oligosaccharide units, whereas the type L protein probably contained 3 or more units.

Sodium ion was shown to stimulate strongly the transport of l -glutamic acid into cells of Bacillus licheniformis 6346 His − . Lithium ion had a slight capacity to replace Na + in this capacity, but K + was without effect. Three of five amino acids tested. l -glutamic acid, l -aspartic acid, and l -alanine, were concentrated against a gradient in the cells. Intracellular pools of these amino acids were extractable with 5% trichloroacetic acid. Pools of l -histidine and l -lysine could not be detected. No evidence of active transport of lysine into cells could be detected, and histidine was taken up in the absence of chloramphenicol but not in its presence. The uptake of glutamic acid by membrane vesicle preparations was strongly stimulated by reduced nicotinamide adenine dinucleotide (NADH) and to a lesser extent by succinate. The presence of phenazine methosulfate increased uptake in the presence of succinate. Either l - or d -lactate and adenosine triphosphate were without effect. None of these compounds stimulated the uptake of glutamic acid by mesosomes, although some mesosome preparations contained separable membrane which was very active. NADH strongly stimulated the uptake of aspartic acid and alanine by membrane vesicles but had only a slight effect on the uptake of histidine and lysine. No evidence of active transport of any of the amino acids into mesosomes could be detected either in the presence or absence of NADH. NADH stimulation of the uptake of glutamic acid by membrane vesicles was destroyed by exposure to light of 360 nm; this inactivation was reversible by vitamin K 2 (5) or K 2 (10) . Sodium ion stimulated transport of glutamic acid by membrane vesicles.

Water-extractable type 6 Klebsiella antigens were separated into a type 6-specific acidic polysaccharide and a neutral polysaccharide. The neutral polymer was devoid of type 6 activity although it was serologically active. The type 6-specific polymer contained fucose, glucose, and mannose, and pyruvic, galacturonic, and possibly glucuronic acids. The neutral polymer contained glucose, galactose, and mannose.

Two strains with mutations in the first structural gene of the histidine operon of Salmonella typhimurium were characterized. (The first structural gene specifies the first enzyme of histidine biosynthesis, phosphoribosyltransferase, which is sensitive to feedback inhibition by histidine.) One mutation, hisG3934 , results in a phosphoribosyltransferase which is no longer sensitive to feedback inhibition by histidine but is instead subject to inhibition by aspartic acid. The other mutation, hisG3935 , allows the histidine operon to be partially repressed by several amino acids, including aspartic acid. Analysis of hisG3935 is consistent with the hypothesis that phosphoribosyltransferase is directly involved in the regulation of the histidine operon.

A tuberculin-active glycopeptide containing eight different amino acids and glucose was isolated from the protoplasm of Mycobacterium tuberculosis . A molecular weight of 4,000 to 5,000 was established by Sephadex gel filtration; other analyses showed a peptide to carbohydrate ratio of 9:1. These observations suggest a tentative composition of 3 to 4 residues of glucose, 12 residues each of aspartic and glutamic acids, 3 residues each of lysine, glycine, and serine, and 1 residue each of arginine, threonine, and alanine.

Biologically active 14 C-labeled purified protein derivative ( 14 C-PPD) has been prepared from the culture filtrates of seven species of mycobacteria, namely Mycobacterium tuberculosis Johnston strain (PPD), M. bovis BCG (PPD-BCG), M. avium (PPD-A), M. kansasii (PPD-Y), M. intracellulare (PPD-B), M. scrofulaceum (PPD-G), and M. fortuitum (PPD-F). These mycobacteria were grown in a culture medium containing a mixture of 14 C-labeled amino acids. The yield and specific radioactivity of the PPD, of the nucleic acid, of the bacterial cells, and of the CO 2 developed during growth have been determined for each of the seven species of mycobacteria. Although the yields of 14 C-PPD antigens differed greatly for the different species of mycobacteria tested, their specific radioactivities were similar. The 14 C-PPD antigens have been used as a means to measure their adsorption to glass. When glass ampoules containing dilute solutions (0.001 mg of PPD per ml) of these PPD antigens (PPD, PPD-BCG, PPD-A, PPD-Y, PPD-G, PPD-B, and PPD-F) were stored for 12 months at 5 C, it was found that they all adsorbed equally well to glass surfaces. In fact, regardless of the origin of the PPD, a loss due to adsorption of about 90% occurred during the first month of storage, and thereafter the PPD content remained practically constant for the rest of the duration of the storage period. The addition of 0.0005% Tween 80 to the PPD solutions effectively reduced the adsorption to glass of most PPD antigens. However, adsorption of PPD-BCG was not quite so effectively prevented, even when the Tween 80 concentration was increased from 0.0005 to 0.0005%.

The specific activities of l -alanine: d -alanine racemase, d -alanine: d -alanine ligase, and the l -alanine, d -glutamic acid, meso -diaminopimelic acid, and d -alanyl- d -alanine adding enzymes were followed during growth of Escherichia coli . The specific activities were nearly independent of the growth phase. d -Alanine: d -alanine ligase was inhibited by d -alanyl- d -alanine, d -cycloserine, glycine, and glycyl-glycine. l -Alanine: d -alanine racemase was found to be sensitive to d -cycloserine, glycine, and glycyl-glycine. The l -alanine adding enzyme was inhibited by glycine and glycyl-glycine.

In a study of the mureins of coryneform bacteria ( Arthrobacter, Brevibacterium, Cellulomonas, Corynebacterium, Erysipelothrix ), 21 threonine-containing strains were found. In several of the strains the amino acid and amino sugar composition of the murein was muramic acid (Mur), glucosamine (GlcNH 2 ), d -Glu, l -Lys, l -Thr, and Ala in a molar ratio of 1:1:1:1:1:4 or 5, and in several other strains it was Mur, GlcNH 2 , d -Glu, l -Lys, l -Thr, Ala, and l -Ser in a molar ratio of 1:1:1:1:1:3:1. The amino acid sequence of the mureins was determined by analyzing the oligopeptides derived from partial acid hydrolysates. It was shown that there were five different murein types. The peptide subunits attached to the muramic acid are the same, namely l -Ala- d -GluNH 2 - l -Lys- d -Ala. In one strain, the α-carboxyl group of d -Glu is substituted by d -alanine amide. The interpeptide bridges of the different types consist of the peptides l -Ala- l -Thr- l -Ala, l -Ala- l -Thr, l -Ala- l -Ala- l -Thr, l -Ala- l -Ala- l -Ala- l -Thr, or l -Ala- l -Thr- l -Ser which are bound through their C-termini ( l -Ala, l -Thr, l -Ser) to the ε-amino group of l -Lys of one peptide subunit and by their N-termini ( l -Ala) to the C-terminal d -Ala of an adjacent peptide subunit. Determination of the N- and C-terminal groups in the mureins showed that about 15 to 30% of the interpeptide bridges are not cross-linked.

The NH 2 -terminal amino acid composition of the soluble and ribosomal proteins from Neurospora crassa mycelia and conidia was determined by the dinitrophenyl method. A nonrandom distribution of NH 2 -terminal amino acids was observed in the complex protein mixtures. Glycine, alanine, and serine accounted for 75% of the NH 2 -terminal amino acids, and glycine appeared most frequently in mature proteins of mycelia. The appearance of phenylalanine as one of the major NH 2 -termini in crude conidial fraction suggests that the composition of proteins may vary in different developmental stages.

Comparative chemical analyses were made of the walls of vegetative cells, heterocysts, and spores, and of the mucilage of Anabaena cylindrica . The wall of the vegetative cell is composed predominantly of amino compounds, with a mannose-rich carbohydrate component comprising only 18% of the dry weight. In contrast, 62% of the heterocyst wall and 41% of the spore wall is carbohydrate. The carbohydrate moieties of the heterocyst wall and spore wall are similar in that the ratio of glucose, mannose, galactose, and xylose is approximately 75:20:3:4 in both walls. It appears that, during the differentiation of a vegetative cell into either a spore or a heterocyst, a glucose-rich wall polysaccharide is produced that is different from the polysaccharide component of the wall of the vegetative cell and of the sheath. In the case of the heterocyst, the wall was estimated to account for approximately 52% of the dry weight of the whole cell.

portion of genetic loci showing variation in outbreeding species (SHAW 1965; HARRIS 1966; and LEWONTIN and HUBBY 1966). Such estimates predict that at least 30 to 40% of genetic loci in*Drosophila, Peromyscus, and man are polymorphic. These predictions have led to a flurry of papers presenting mathematical models which show that, contrary to earlier dogma (discussed by KIMURA and CROW 1964; VAN VALEN 1963), populations could theoretically sustain this degree of variation through balanced polymorphism (KING 1967; MILKMAN 1967; SVED, REED, and BODMER 1967). If heterozygote superiority is the responsible factor for maintaining a high degree of genetic polymorphism, one presumes that the increase in number of proteins associated with heterozygosity offers, in general, the selective advantage. In contrast to the attention which polymorphism has received as a mechanism for maintaining an increased number of proteins in heterozygotes, a second type of protein multiplicity, while widely recognized, has received much less attention in terms of its evolutionary significance. It is illustrated by isozyme patterns showing multiplicity of enzymes shared by every individual in the mating population (the lactic dehydrogenase isozymes of the human are an example). Preliminary estimates indicate that this type of protein diversity, probably arising primarily from gene duplication, is very common. For example, we have recently reported (BREWER and SING 1968a) that ten of sixteen randomly selected human red cell enzymes show isozyme multiplicity common to all individuals. We suggest that this type of protein diversity may make a significant contribution to the total protein diversity available to the individual. In a cross fertilizing species such as man, this could be an alternative strategy to allelic variation (FINCHAM 1966), but it may be of prime importance in a species characterized by obligatory inbreeding, as for example, in the self-pollinator, wheat. This paper presents initial studies of the correlation between protein multiplicity, as measured by the zymogram technique, and certain biological characteristics of hexaploid wheat and its tetraploid and diploid progenitor species. The isozyme data reported here were collected to obtain information on three This investigation was supported by contract AT(11 1) 1152 Atomic Energy Commission, USPHS grant AM 09381

Groups of young albino mice were fed continuously four different types of diets and were compared with regard to (1) rate of weight gain; (2) resistance to experimental bacterial infections. The protein content of the four diets was as follows: (a) pellets: a minimum of 21 per cent "crude" protein (according to the manufacturer); (b) diet 20 C: 20 per cent casein; (c) diet 8 C: 8 per cent casein; (d) diet 8 C + AA: 8 per cent casein supplemented with 12 per cent of a mixture of essential amino acids. All diets provided an adequate supply of minerals and vitamins. They were administered ad lib. Three strains of pathogens virulent for mice were used for the infection tests, namely: Staphylococcus aureus, Mycobacterium fortuitum, and Mycobacterium tuberculosis bovis. The bacteria were injected by the intravenous route. The experimental regimens were begun at different times before infection, and were continued until death of the animal, or until termination of the experiment. It was found that mice on the 8 C diet exhibited much greater susceptibility to infection than did mice on the 20 C diet; mice receiving pellets were intermediate between these two groups. The infection-enhancing effect of the 8 C diet could be entirely corrected by amino acid supplementation (diet 8 C + AA). Indeed, mice fed diet 8 C + AA proved the most resistant to infection. The fact that animals fed pellets (which contain a minimum of 21 per cent protein) consistently died faster following infection than did animals fed diets 20 C or 8 C + AA suggests that qualitative characteristics of the protein in the regimen are as important as the quantity of protein fed in determining susceptibility to infection. The differences in susceptibility exhibited by the mice on the four experimental diets were the same whatever the species of bacterial pathogen used for the infection test, the size of the infective dose, and the duration of the disease. There was no apparent relation between the effects of the diets on the weight curves of the animals, and on resistance to infection. Mice on diet 8 C (which were most susceptible) gained weight as rapidly as those on 20 C and more rapidly than those fed 8 C + AA (which were most resistant). All the tests reported in the present paper were carried out with young mice, which were placed on experimental diets within 1 to 2 weeks after weaning. Preliminary experiments suggest that the relation between dietary factors and susceptibility to infection was more difficult to bring out in older animals. There was evidence also that this relation was most apparent during the first weeks that the animals were fed the experimental diets, and became less striking after several weeks.

The paper contains a description of the fine structure of the thyroid gland of the normal rat. The follicular colloid, a homogeneous substance of faintly granular texture, is bounded by cuboidal or low columnar epithelial cells. Numerous pleomorphic microvilli, often permeated by small vesicles extend from the apices of the epithelial cells into the colloid. Many small, membrane-limited vesicles lie in the superficial cytoplasmic layer just below the apical plasmalemma. The ergastoplasmic sacs of the follicular cells are dilated and contain a substance resembling colloid. They are of irregular outline, and the larger sacs tend to be located in the base of the cells. The Golgi apparatus lies in the vicinity of the nucleus and consists primarily of numerous small, membrane-bound droplets with a homogeneous content. Droplets, similar to the Golgi vesicles but larger, lie in the same vicinity and are tentatively identified as colloid droplets. The colloid droplets contain an extremely fine, dense particulate material. Other droplets with a denser, more heterogenous content are also present. Both the follicular cells and the perifollicular capillaries are bounded by a continuous basement membrane. The capillary endothelium is in certain regions extremely attenuated and is pierced by numerous patent pores, 450 A in diameter. The marked similarity between the presumptive colloid droplets and vesicles of the Golgi apparatus suggests that the droplets arise from this organelle. On morphological grounds alone no relation can be established between any of the organelles of the follicular cell and the process of colloid resorption.

Caulerpa racemosa var. macrophysa, C. racemosa var cornyphora and C. scalpelliformis are analyzed by quantitative paper chromatographic technique for their amino acid contents in proteins, peptides and free state. It is found that no appreciable variation occurs in the quality of amino acid make up in these algae; but quantitative differences are apparent in them. Moreover, both qualitative and quantitative variations occur in the amino acid make up of the peptides, and in the free state. The results are compared with those of the other investigations.

Most investigators have used excised tissue, cellfree preparations, or whole plants for studies of metabolism in angiosperms. Each of these systems represents a different state of the cells or tissues. Another state of plant cells, that of exponential growth, has been obtained by tissue culture techniques (2, 5). Studies of the metabolism of cells growing exponentially would provide information which is complementary to that already available for plants. Such studies would allow direct comparison of metabolism of higher plant cells with that of microorganisms also growing exponentially. When specific information is not available, it is generally assumed that the metabolism in plant cells is the same as that in micro-organisms. This assumption may be tested using exponentially growing plant cells. For use in studies of metabolism a culture system in which plant cells are growing exponentially should have several features. The media used should have a known composition. The tissue should be grown in suspension as very small clumps or single cells. The use of media of known composition makes it possible to manipulate the nutrients at will and to avoid the presence of unrecognized compounds which occur in media supplemented with natural extracts. The growth of tissues as suspensions of single cells or small clumps of cells prevents the formation of the diffusion gradients which occur in larger pieces of tissue. Rapid agitation of the suspension keeps the cells in equilibrium with the medium and the medium in equilibrium with its gaseous atmosphere. Such desirable features can now be found in at least 2 plant tissue cultures. These are cultures established from Paul's Scarlet Rose by Dr. W. Tulecke (Tulecke, private communication) and from Nicotiana tabaccumn var. Xanthi by Dr. P. Filner (22). Methods established for growth of microbial cultures have been successfully adapted to the culture of plant cells. For example, plant tissues have been maintained on solid media as callus. Single plant cells have been plated in agar to yield visible colonies (8). Also, plant tissues have been maintained as suspensions of cells and clumps in liquid media of known or unknown composition (2) in shake flasks or large carboys (29). The close parallels in methods of growth suggest that methods developed to study the nietabolism of microbial cells in culture might also be readily adapted to the study of plant cell metabolism. Several studies (10, 18, 34) of plant metabolism have employed the isotope competition method (25). This paper is to report the application of the isotope competition method to the study of amino acid biosynthesis in suspensions of plant cells growing exponentially in media of known composition.

The preceding paper (10) showed that the growth of Escherichia coli is slowed, without killing, at 40 to 45 C, and that in the several strains tested the cause is a decrease in the activity of homoserine trans-succinylase. These temperatures are now shown to inhibit the enzyme directly, in crude extracts and after partial purification. The effect is rapid and is immediately reversible, unlike the progressive and slowly reversible changes of conventional denaturation.

The experiments presented in this paper support earlier evidence that ribosomes are released from polysomes when they encounter a codon for which no charged transfer ribonucleic acid is available. However, it is further shown that these ribosomes then reinitiate and resume translation. The size and the level of polysomes during deprival of an amino acid is a function of the frequency with which that particular amino acid appears in cellular proteins. Polysomes from starved cells are more stable than those from growing cells, and, moreover, polysomes from starved relaxed strains are more stable than those from starved stringent strains.

The paper describes a new and gentle procedure for isolating “structural” phospholipoproteins from organisms and the first such isolation from a procaryotic microbe, Hydrogenomonas facilis . The amino acid composition of its protein moiety resembles that of “structural protein” from other sources. Although disc gel electrophoresis has shown the protein to be heterogeneous, this is attributed to aggregation.

1. beta-Amylase obtained by acidic extraction of soya-bean flour was purified by ammonium sulphate precipitation, followed by chromatography on calcium phosphate, diethylaminoethylcellulose, Sephadex G-25 and carboxymethylcellulose. 2. The homogeneity of the pure enzyme was established by criteria such as ultracentrifugation and electrophoresis on paper and in polyacrylamide gel. 3. The pure enzyme had a nitrogen content of 16.3%, its extinction coefficient, E(1%) (1cm.), at 280mmu was 17.3 and its specific activity/mg. of enzyme was 880 amylase units. 4. The molecular weight of the pure enzyme was determined as 61700 and its isoelectric point was pH5.85. 5. Preliminary examinations indicated that glutamic acid formed the N-terminus and glycine the C-terminus. 6. The amino acid content of the pure enzyme was established, one molecule consisting of 617 amino acid residues. 7. The pH optimum for pure soya-bean beta-amylase is in the range 5-6. Pretreatment of the enzyme at pH3-5 decreases enzyme activity, whereas at pH6-9 it is not affected.