Smallest Functional Unit Research Articles

Loops and secondary structure mimetics: development and applications in basic science and rational drug design.

One goal of protein design and structural biochemistry is the reduction of complex molecules to small functional units that are amenable to high resolution analysis and rapid modification. We have developed a variety of small molecules which biochemically and biologically mimic the combining sites of proteins of the immunoglobulin superfamily. The chemical and biological properties of peptide mimetics suggest that these analogs can be used as indicators for new pharmaceutical agents. Mimetics are powerful tools for the study of molecular recognition since they are small in size, maintain solubility in physiologic fluids and are amenable to detailed structural studies. As such, they represent a step toward the rational design of low molecular weight non-peptide pharmaceutical agents devoid of some of the shortcomings of conventional peptides. Here we discuss the rationale and approaches for the development of these molecules, and their current and future applications.

A review of techniques employed to estimate the number of motor units in a muscle

Being the smallest functional units under neural control, motor units play an integral role in muscle physiology. However, at the present time, there does not exist any widely accepted technique for quantifying or estimating the number of motor units in a muscle. Specifically, the existing techniques are the increment-counting technique, a technique based on spike-triggered averaging, and a macro-EMG based technique which vary in invasiveness from noninvasive to highly invasive, respectively. We discuss each of these techniques, along with their associated shortcomings, in detail.

Control of the Utah/MIT dextrous hand: Hardware and software hierarchy

AbstractThis article describes a hardware and software hierarchy for the control of manipulation with the Utah/MIT Dextrous Hand (UMDH). Control algorithms execute within a multiprocessing, shared memory computer environment partitioned into a three tiered hierarchy. Joint level servo algorithms are executed at the lowest level, trajectory generation and communication at the middle level and at the highest level, a new programming language called HPL (Hand Programming Language) is implemented. A functional approach to the control of manipulation is described and the functional philosophy is embedded within HPL. Tasks are specified by the concatenation and linear combination of small functional units called motor primitives. HPL provides an abstraction of the process of manipulation through its use of motor primitives. Hand control programs can be written and easily refined within the HPL programming environment and the resulting programs act as a conveniently annotated description of manipulation tasks.

An experimental approach to testing modular evolution: directed replacement of alpha-helices in a bacterial protein.

We have used oligonucleotide site-directed mutagenesis to ask whether certain structural motifs in proteins are determined mainly by local interactions among amino acids. Multiple consecutive amino acids in three alpha-helices in the alkaline phosphatase (EC 3.1.3.1) of Escherichia coli have been replaced with helical sequences from four other sources. Altogether, 12 distinct helical replacements were created, 9 of which retain enzymatic activity. Most short stretches of helical sequence can be replaced with unrelated helical sequences without eliminating enzyme activity. Replacements of the carboxyl half of an alpha-helix are less harmful than those of the amino half, and the two together are synergistic rather than additive. These results are consistent with the hypothesis that proteins originally evolved by the assembly of small functional folding units.

Solubilized bovine striatal D2 dopamine receptor regulated by sodium ion and guanine nucleotides

D2 dopamine receptor was solubilized in a sensitive form for sodium ion and guanine nucleotides with a zwitterionic detergent, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), from bovine striatum. The presence of sodium ion in the solubilization mixture markedly increased the yield. The solubilized D2 receptor possessed characteristics of : (a) the site was localized in the striatum but not in the cerebellum; (b) high affinity to nanomolar concentrations of [3H]-spiperone; (c) displacing potencies of various dopamine agonists and antagonists were similar to those of membrane-bound receptors; (d) guanine nucleotides decreased the affinity of soluble receptor for dopamine agonists, but not for antagonists ; (e) negative heterotropic effect of guanine nucleotides was seen only when sodium ion was present in the assay mixture. The solubilized receptor complex was eluted on Sepharose CL-4B column chromatography as a large molecule which had a Stokes radius about 90 Å. Radiation inactivation of membrane-bound D2 dopamine receptor revealed the presence of large (120 0 -1400 K) and small (80 - 100 K) functional units. Therefore, the oligomeric complex between the D2 dopamine receptor and GTP binding protein probably remains intact throughout the solubilization procedure.

Pyridine nucleotide transhydrogenase from Azotobacter vinelandii. Improved purification, physical properties and subunit arrangement in purified polymers.

1. Pyridine nucleotide transhydrogenase from Azotobacter vinelandii was purified with a scaled-up procedure. In a typical purification 500 ml cell-free extract from 200 g cells is loaded on an Ado-2',5'-P2--Sepharose 4B affinity column (20 ml bed volume). After washing, the enzyme is desorbed with 2'AMP at neutral pH and further purified by Sephadex G-200 gel chromatography. The enzyme (10--12 mg) is obtained in 40--60% yield and is homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. 2. The homogeneity of the purified enzyme is also apparent from electron microscopy studies, where the enzyme appears as a polydisperse set of polymers without contaminating structures and from fluorescence lifetime studies by the method of single-photon counting. The flavin fluorescence appears to decay with a single lifetime tau = 2.5 ns. The polymeric nature of transhydrogenase can be aptly demonstrated by density gradient centrifugation in the presence of KBr. After centrifuging for 50 h at 160 000 X g and 10 degrees C the enzyme is concentrated in a narrow fluorescent band with buoyant density rho b = 1.305 g cm-3. 3. The arrangement of subunits in the transhydrogenase polymer has been derived from optical diffraction studies of electron micrographs. The polymers are built up from a linear assembly of tetramers. Four subunits are placed in a rhomb with sides of 13.5 mm and an angle of 45 degrees (135 degrees) between the sides. A second tetramer is located staggered on top of the first one. Since a variety of other studies have indicated that the polymers dissociate into octamers under alkaline conditions [Voordouw, G. et al. (1979 Eur. J. Biochem. 98,447--454] we conclude that this smallest functional unit is build up from two tetramers.

Physical properties of small functional units obtained from gastropod mollusc haemocyanins by tryptic digestion.

The haemocyanins of gastropod molluscs are giant proteins of mol.wt. approx. 9000000 whose function is the transport of oxygen in the blood. It is well established that the functional unit, which binds one molecule of 02, is a pair of copper atoms surrounded by a folded polypeptide chain to form a compact domain of mol.wt. approx. 50000 (Van Breemen et al., 1977). However, treatment of the protein with such reagents as 6~-guanidinium chloride or sodium dodecyl sulphate fails to produce a subunit of this size, but appears to reveal a minimum polypeptide chain of molecular weight in the region of 300000 (Wood P Brouwer & Kuiper, 1973). Functional subunits of mol.wt. 50000 have so far only been obtained by limited proteolytic digestion of the native protein (Pearson & Wood, 1974). Electron-microscopic studies have revealed necklace-like strings of linked domains and give valuable clues as to how the native molecule is constructed (Van Breeman et al., 1977). Moreover work on the limited proteolytic digestion of Helix pomatia 8-haemocyanin and Lymnaea stagnalis haemocyanin suggests that the domains may differ from one another in their structure and properties (Gielens et al., 1977; Wood & Mosby, 1977). In the present work we have studied the effect of trypsin hydrolysis on the haemocyanins from a marine gastropod, Buccinum undatum, and from a freshwater gastropod, Lymnaea stagnalis, which are known to differ in their oxygen-binding properties as well as in amino acid and carbohydrate composition (Hall & Wood, 1976; Hall et al., 1977). It is hoped that studies on the fundamental building-blocks of the molecule will

METABOLIC PORTOSYSTEMIC SHUNTS“ IN ORNITHINE TRANS-CARBAMYLASE DEFICIENCY?

Females with ornithine transcarbamylase (OTC) deficiency, an X-linked disorder, are mosaic in that they have hepatocytes with almost no enzyme activity and hepatocytes with normal enzyme activity. The normal and abnormal cells occur in clusters since cells in an area tend to be the progeny of the same parent cell. Marked variation in OTC was found in small liver biopsies from a female with OTC deficiency, probably because the samples included clusters of normal or abnormal cells. OTC activity was measured in 10 approx. 5 mg specimens of liver from a single surgical biopsy from a 7 year old girl with OTC def. (two experiments and control autopsy liver (three experiments). The OTC activity in the patient varied 10-40 fold in the two experiments with a coefficient of variation of 76 and 97 percent (control 20,21 and 26 percent). Thus OTC activity in a small biopsy in females with OTC def. may not be representative of the entire liver. The OTC activity in several bits of liver from our patient was very low suggesting that clusters of abnormal cells may often involve a major portion of a hepatic lobule. If so, blood passing through these areas would in effect be passing through a “metabolic shunt.” These shunts may, in part, account for the hyperammonemia in this disorder. The notion that an X-linked disorder can produce disease in heterozygotes by affecting small functional units could apply to other disorders such as nephrogenic diabetes insipidus or vitamin D dependent rickets.

Scanning processes and sentence recognition

This report summarizes an experiment designed to study the disruptive effects on immediate recall of interfering with the ability to scan ahead. Sentences were printed on paper tape which was pulled through a window, the size of which could be controlled. There were two exposure widths, three rates of tape movement, and three types of material which varied in length: equational and transitive sentences as well as random strings of words. Errors increase as the exposure width is decreased, and the magnitude of the disruptive effect is most marked for the longer sentences and the faster rate of presentation. A possible explanation is advanced emphasizing the likelihood that Ss store information in a sensory register and scan that register in order to segment sentences into phrases, phrases into words, and words into smaller functional units. As one limits exposure width, one interferes with the transfer of information into the sensory register and with the processing of information after it is stored in the register.

An information retrieval system using keyword dialog

This paper describes an approach to information retrieval through keyword dialog. The problems of using English for information retrieval are delineated. The querying capability of an existing system using the keyword dialog is illustrated with actual computer output. Some of the major advantages of this approach are: it reduces ambiguities in requests; it allows the user to think in smaller functional units; it allows the user to edit previous requests. The high usage during a 6-month trial demonstrated that this approach is a viable alternative to using uncontrolled English.

α-Keto Acid Dehydrogenase Complexes: XII. EFFECTS OF ACETYLATION ON THE ACTIVITY AND STRUCTURE OF THE DIHYDROLIPOYL TRANSACETYLASE OF ESCHERICHIA COLI

Dihydrolipoyl transacetylase, one of the three enzymes comprising the Escherichia coli pyruvate dehydrogenase complex, consists of 24 identical polypeptide chains that are linked by noncovalent bonds. There appear to be 3 tyrosyl residues per chain of molecular weight about 40,000. Treatment of the transacetylase with increasing amounts of N-acetylimidazole resulted in gradual loss of enzymatic activity and concomitant O-acetylation of tyrosyl residues. This treatment also caused the enzyme to dissociate into subunits. The partially inactivated transacetylase was separated into two classes of subunits, with sedimentation coefficients of 4 to 7 S and 11 to 15 S, by gel filtration and by sucrose density gradient centrifugation. The former class of subunits contained a species with a molecular weight of about 120,000. Both classes of subunits exhibited approximately the same specific activity. It appears that inactivation and dissociation of the transacetylase are parallel, rather than sequential, phenomena. The various subunits retained the capacity to combine with pyruvate dehydrogenase and dihydrolipoyl dehydrogenase, forming complexes capable of catalyzing the oxidation of pyruvate to acetyl coenzyme A. The smallest functional unit had a molecular weight of about 500,000.

Filament ultrastructure and organization in vertebrate smooth muscle. Contraction hypothesis based on localization of actin and myosin.

Using a variety of preparative techniques for electron microscopy, we have obtained evidence for the disposition of actin and myosin in vertebrate smooth muscle. All longitudinal myofilaments seen in sections appear to be actin. Previous reports of two types of longitudinal filaments in sections are accounted for by technical factors, and by differentiated areas of opacity along individual filaments. Dense bodies with actin emerging from both ends have been identified in homogenates, and resemble Z discs from skeletal muscle (Huxley, 1963). In sections, short, dark-staining lateral filaments 15-25 A in diameter link adjacent actin filaments within dense bodies and in membrane dense pataches. They appear homologous with Z-disc filaments. Similar lateral filaments connect actin to plasma membrane. Dense bodies and dense patches, therefore, are attachment points and denote units analogous to sarcomeres. In glycerinated, methacrylate-embedded sections, lateral processes different in length and staining characteristics from lateral filaments in dense bodies exist at intervals along actin filaments. These processes are about 30 A wide and resemble heavy meromyosin from skeletal muscle. They also resemble heads of whole molecules of myosin in negatively stained material from gizzard homogenates. Intact single myosin molecules and dimers have been found, both free and attached to actin, even in media of very low ionic strength. Myosin can, therefore, exist in relatively disaggregated form. Models of the contraction mechanism of smooth muscle are proposed. The unique features are: (1) Myosin exists as small functional units. (2) Movement occurs by interdigitation and sliding of actin filaments.

STUDIES ON SPECIES OF OIDIODENDRON, HELICODENDRON, AND STACHYBOTRYS FROM SOIL

Oidiodendron nigrum Robak was recorded for the first time from soil. The method of conidial production was examined and it was found that thick-walled endospores were produced within the parental hyphal walls rather than a simple fragmenting and rounding-off of sporiferous hyphae. An isolate of Helicodendron tubulosum from soil was found to differ from previously described isolates in the production of a second phialospore-type conidial stage and in the ability of mature helicoid spores to fragment at maturity into a number of smaller functional units. Isolates corresponding to Stachybotrys cylindrospora Jensen were examined and found to be distinct from Stachybotrys atra with which it had previously been regarded as synonymous.