Small Molecular Weight Proteins Research Articles

CHARACTERIZATION OF THE CHLOROGENIC ACID BINDING FRACTION IN LEAF PROTEIN CONCENTRATION

ABSTRACTLeaf protein concentrate (LPC) haa been shown to have the phenolic acid, chlorogenic acid, bound to its protein molecules. Bound chlorogenie acid creates off color problems in foods containing LPC as an ingredient and also possibly lowers the digestibility of LPC by inhibiting the rate of trypsin attack on the protein. A buffer, containing 2.5% NaCl‐1 mM mercaptoethanol‐40 mM phosphate, effectively removed 69% of the chiorogenic acid from LPC. LPC, with the majority of its chlorogenic acid removed, had an enhanced in vitro protein digestibility and an amino acid pattern very similar to that of the original LPC. A series of small molecular weight proteins were found to be the chlorogenie acid binding fraction in LPC.

Fractionation of fowl immunoglobulins

Serum, Na2SO4-precipitated serum immunoglobulins and bile from 12-week-old fowls, and serum from day-old chicks, were fractionated by Sephadex G-200 gel filtration, DEAE Sephadex A-50 ion exchange chromatography and ultracentrifugation through 10-40 per cent sucrose gradients. Elution of IgM, IgG, IgA and albumin was monitored by examination of fractions in agar gel diffusion against antisera specific to these proteins. Serum and bile from 12-week-old fowls contained IgM and IgA in two molecular sizes and a single molecular size of IgG. Day-old chick serum contained IgM estimated to be 7S, a polymerised form of IgG in addition to the normal 7S component, and a small molecular weight protein antigenically related to IgA. Most of the albumin in bile was of lower molecular weight than serum albumin, while heavy forms of albumin were detected in ultracentrifugation of bile and day-old chick serum.

Partial purification and characterization of a glutamate-binding membrane glycoprotein from rat brain

Summary A 200-fold purification of a synaptic membrane glutamate-binding protein was achieved by a combination of affinity batch separation on glutamate-loaded glass fiber and affinity chromatography on concanavalin A sepharose. Analytical gel electrophoresis of this fraction in Triton X-100 revealed a predominant acidic, small-molecular weight protein species exhibiting all of the glutamate binding activity and having a molecular weight of 13,800 as estimated by SDS gel electrophoresis. This purified protein did not show any glutamate dehydrogenase, glutamate decarboxylase, or glutamine synthetase activity, and it differed from the glutamate transport system in many of its characteristics. On the other hand, its interaction with L-glutamate and other neuroexcitatory amino acids resembled that of the physiologic receptor for L-glutamic acid.

Antigens and Antigenic Variability of the African Trypanosomes*†

SYNOPSIS. The recent literature on the physical, chemical and biological characterization of antigens from the African trypanosomes is reviewed. The antigens are divided into three major groups: a) variant‐specific antigens, b) common antigens, and c) host‐like antigens. The variant‐specific antigen(s) are relatively small molecular weight proteins located on the surface of the trypanosome, and are involved in protection and agglutination. It is suggested that there is more than a single variant‐specific antigen on the cell surface. In contrast, the common antigens are internal or somatic antigens which are believed to have structural or enzymatic functions but are not involved in either protection or agglutination. In addition, evidence is also presented which suggests that there are host‐like antigens within the surface coat and/or membranes of the trypanosomes. The importance of these three groups of antigens, and the mechanis(s) of antigenic variation are discussed in relationship to the immune response of the host to the African trypanosomes. Several possible approaches for future investigations are described.

Protein synthesis in phenotypically different, single neurons of Aplysia

Different molecular weight classes of proteins from single phenotypically different neuron somas and their subcellular fractions were analyzed in terms of the relative concentration and relative labeling using sodium dodecyl sulfate (SDS) polyacrylamide microgel electrophoresis. A 12,000 (12K) molecular weight class of protein found in low relative concentration but highly labeled was shown to be present in some endogenously firing neurons, L3, L6, and L11, but not in others such as L4. A specific 12K class of protein has been found to be common to all neurosecretory neurons, R15, R14 and R3–13, studied. A high relative concentration of 6–9K class of protein in R15 and bag cells and a highly labeled 6–9K protein shown to be in R14 and R3–13 have been correlated with neurosecretion. In subcellular fractionation studies a specific 12K class of proteins was found in the membrane fraction of neuron L11. Several proteins, which have molecular weights similar to filarin (77K), tubulin (58–55K) and actin (43K) have been shown to be common to all neurons studied. Hence this study has demonstrated protein specificity in single phenotypically different neurons. Suggestive correlations of specifically translated small molecular weight proteins with neurosecretion in neurons R3–15 were made.

Properties of a thermosensitive asporogenous filamentous mutant of Bacillus megaterium.

Mutant TH14 of Bacillus megaterium ATCC 19213 is thermosensitive and defective in cell-division septation and spore formation at the restrictive temperature (39 C). As a consequence, the mutant forms multinucleate aseptate filaments and is asporogenic. The mutation does not result in any qualitative compositional changes in extractable membrane proteins. At the restrictive temperature, the mutant membrane has a reduced content of a small molecular weight protein(s). A membrane protein(s) with a molecular weight of nearly 80,000 appears to be partially derepressed in the mutant grown at the restrictive temperature. In addition, numerous unidentified spherical inclusions of fairly uniform size (diameter approximately 100 nm) are present in the cytoplasm at the restrictive temperature. They are especially concentrated at only one pole of each filament. Filamentous growth of the mutant is less sensitive to penicillin than growth in the rod form. Growth in either form is equally sensitive to d-cycloserine at the concentrations used for selection of the mutant. Temperature shift-up experiments suggest that one to two rounds of deoxyribonucleic acid (DNA) replication occur before the phenotypic expression of the mutation occurs. The septations after these replication events can be either two-division septations or a single-division septation plus a subsequent sporulation septation. This conclusion, coupled with previously reported work, supports the hypothesis that the early stages of sporulation represent a modified cell division.

Immunoreactive growth hormone in plasma and urine in long term insulin treated diabetics with clinical diabetic nephropathy.

ABSTRACT Plasma immunoreactive growth hormone (IRHGH) was measured serially together with 24 hour urinary immunoreactive growth hormone (IRHGH) in 25 long term insulin treated diabetics with clinical diabetic nephropathy (defined by urinary albumin above 25 mg/24 h). The mean plasma IRHGH was significantly higher than in a comparable group of diabetics without increased urinary albumin (0.05 > P > 0.02). The urinary IRHGH increased from near normal values in those patients with a normal creatinine clearance to 100 times the normal urinary IRHGH in patients with creatinine clearance of less than 20 ml/min. Two other small molecular weight proteins (β2-microglobulin and lysozyme) were also measured in the blood and urine in some of the patients. IRHGH, β2-microglobulin and lysozyme clearances increased in parallel as the creatinine clearance decreased. This study further supports the theory that growth hormone is filtered by the glomerulus and reabsorbed for the major part in the proximal renal tubule. The increased plasma and urinary IRHGH seen in diabetic nephropathy seems to be a consequence of the kidney damage rather than its cause.

Yeast Pro-proteinase C Part IV

Activation of pro-proteinase C by yeast proteinase A was examined under controlled conditions. Maximum activation occurred at pH 3.5 and 0°C, releasing a cationic protein. The active enzyme and the protein was separated and chemically analyzed. The same N-terminal amino acid, lysine, was found in both the active enzyme and proenzyme. Amino acid analysis revealed that the released protein is a protein of small molecular weight about 19, 000 containing one SH-group and one disulfide bond. These results strongly suggested that the protein would correspond to the cationic subunit of the proenzyme. In both activation processes by denaturant and proteinase A, a decrease of β-structure was found as determined from ORD and CD measurements. All of these results supported the idea that activation of the proenzyme occurred by denaturant- or enzyme-modification of the inhibitor protein, followed by demasking of the active site.

Purification of animal neurotoxins. Isolation and characterization of four neurotoxins from two different sources of Naja haje venom.

Using the same method of purification of small molecular weight proteins worked out to obtain pure scorpion neurotoxins, four neurotoxins have been isolated from the venom of the cobra Naja haje. N. haje venom from the Miami Serpentarium contains two neurotoxins (I and II) whereas the venom of the same species obtained from the Institut Pasteur contains three neurotoxins (I, II′ and III). Toxins I, II and II′ consist of 61 amino acid residues and toxin III of 71. All are basic proteins containing four or five disulphide bridges. It is likely that the difference observed in neurotoxin composition of N. haje venoms from two different sources has a taxonomical counterpart, each venom originating from a different sub‐species.

Two specific azodye-carcinogen-binding proteins of the rat liver: The identity of amino acid residues which bind the azodye

Two soluble liver proteins in the rat have a high affinity of binding for metabolites of azodye carcinogens. One protein is referred to as the basic azodye-binding protein (mol. wt. 45 000, p I 8.4) and the other as the small molecular weight protein (mol. wt. 13 800). A study has been made of the amino acid residues which bind the azodye metabolites in these two proteins. Azodye-bound components were released from these proteins by Pronase digestion followed by refluxing in alkali-ethanol. They were then separated by ‘fingerprinting’. 35S-Labelled amino acids were administered to rats together with azodye. After the administration of L-[ 35S]methionine, azodye-bound components from both types of protein were radioactive: after the administration L-[ 35S]cysteine, azodye components from the basic protein only were radioactive. Thus, since methionine is an essential amino acid, in the basic azodye-binding protein azodye metabolites bind to cysteinyl residues and in the small molecular weight azodye-binding protein, binding is to methionyl residues.

Mycotoxins of entomophthoraceous fungi

Among the four entomophthoraceous species, Entomophthora coronata, E. apiculata, E. thaxteriana, and E. virulenta, only the first two species produced culture filtrates that were toxic when inoculated into the hemocoels of the larvae of seven insect species. The culture filtrates, however, produced no apparent toxicity when fed to the larvae of Galleria mellonella. The fungi produced mycotoxins in all media suitable for their growth in vitro and in vivo. The mycotoxins produced by the two fungi had similar properties and appeared to be identical, but E. coronata produced much more toxin than E. apiculata in the same period of time. More toxin were produced by both fungi in shake than in stationary cultures. The median effective doses (ED50) of the culture filtrates (96-hour cultures) were estimated from data. The mycotoxin was not chemically identified, but tests indicated that it was a protein of small molecular weight. The mycotoxin caused a characteristic blackening sign in inoculated larva of Galleria mellonella; it also inhibited metamorphosis and affected larval feeding. These signs and symptoms varied among the seven insect species. The mycotoxin also inhibited bacterial growth in dead G. mellonella larva. In Galleria mellonlela larva, the mycotoxin apparently altered the staining properties of the hemolymph and caused the clumping and coagulation of the hemocytes. Silk gland cells also showed pathologies, but other tissues and organs appeared to be unaffected. Blood color changes and clumping of the hemocytes did not occur in fungus-infected larvae.

Renal Clearance of Small Molecular Weight Proteins and Enzymes [Summary

Renal Clearance of Small Molecular Weight Proteins and Enzymes [<i>Summary</i>

Purification of Dihydrofolate Reductase via Amethopterin-Aminoethyl Starch

Abstract Amethopterin has been linked covalently, via a carbodiimide-promoted synthesis, to the aminoethyl derivative of soluble starch. The resulting material (mol wt g 300,000) has been used in a novel procedure for purification of chicken liver dihydrofolate reductase (mol wt 22,000). Amethopterin-aminoethyl starch was admixed with the crude enzyme at pH 6.0 and the resulting high molecular weight, enzyme-inhibitor complex was separated from small molecular weight proteins by filtration through Sephadex G-100. The complex was then dissociated by changing the pH to 8 (in the presence of added dihydrofolate), and the highly purified enzyme was recovered by refiltration through Sephadex.

The ultrastructural basis of alveolar-capillary membrane permeability to peroxidase used as a tracer.

The permeability of the alveolar-capillary membrane to a small molecular weight protein, horseradish peroxidase (HRP), was investigated by means of ultrastructural cytochemistry. Mice were injected intravenously with HRP and sacrificed at varying intervals. Experiments with intranasally instilled HRP were also carried out. The tissue was fixed in formaldehyde-glutaraldehyde fixative. Frozen sections were cut, incubated in Graham and Karnovsky's medium for demonstrating HRP activity, postfixed in OsO4, and processed for electron microscopy. 90 sec after injection, HRP had passed through endothelial junctions into underlying basement membranes, but was stopped from entering the alveolar space by zonulae occludentes between epithelial cells. HRP was demonstrated in pinocytotic vesicles of both endothelial and epithelial cells, but the role of these vesicles in net protein transport appeared to be minimal. Intranasally instilled HRP was similarly prevented from permeating the underlying basement membrane by epithelial zonulae occludentes. Pulmonary endothelial intercellular clefts stained with uranyl acetate appeared to contain maculae occludentes rather than zonulae occludentes. HRP did not alter the ultrastructure of these junctions.

Cellular Activities in Hypersensitive Reactions

Summary A basic protein of small molecular weight isolated from bovine nerve tissue induces allergic encephalomyelitis in guinea pigs when injected in small amount in Freund's adjuvant. This protein has been shown to reside in the myelin sheaths of the animal of origin as well as of guinea pigs and man. The responses which follow the administration of nerve tissue to guinea pigs include a specific reactivity of lymphocytes of nodes and spleen for this purified antigen and delayed skin reactivity to it, in the absence of circulating antibodies detectable by ordinary serologic methods. The relationship of lymphocytic and skin reactivity and of disease induction to a purified protein antigen of the myelin sheath strongly suggests a contingency of the lesions upon the reactivity of the cells. However, the intervention of cell-bound or cell-produced antibodies cannot yet be excluded as a basis for the pathologic process.

INCORPORATION OF S 35 INTO RIBOSOMAL PROTEINS OF ESCHERICHIA COLI.

Duerre, John A. (University of North Dakota, Grand Forks). Incorporation of S(35) into ribosomal proteins of Escherichia coli. J. Bacteriol. 88:130-136. 1964.-The rate of incorporation of S(35) was followed in exponentially growing cultures of Escherichia coli. Within a few seconds, the isotope was incorporated into proteins, appearing first in or on the ribosomal particles. Ribosomal proteins from these particles were fractionated into molecular weight classes by gel filtration. The radioactivity from S(35)O(4) (=) appeared in the proteins of large molecular weight at a faster rate than in those of small molecular weight. This radioactivity could be displaced from the large molecular weight class with nonradioactive sulfate more rapidly than from the small class. The large molecular weight protein could also be distinguished from the small molecular weight proteins on basis of their higher sulfur content, which corresponds to that of soluble proteins.

Immunologic studies of the proteins of human synovial fluid

AbstractSynovial fluid and serum were compared by immunoelectrophoresis. The quantity of the large molecular weight proteins (beta 2 macroglobulin, beta lipoprotein, alpha 2 macroglobulin) was markedly reduced in normal synovial fluid. Small molecular weight proteins were present in all synovial fluids. Using an antiserum to synovial fluid, a protein (beta 2F) was detected in inflammatory synovial fluids. This protein appears to be a degradation product of fibrinogen, and was not present in either normal synovial fluid or serum.